Immunological

techniques

Wei Chen Associate professorInstitute of Immunology

E-mailchenwei566zjueducnhttpmypagezjueducn566 8888

Objectives

1048698 To know the main laboratory detection method using antibody

To know how to isolate the specific lymphocyte population

1048698 Be able to describe how to detect the change of human immune function after bacteria or virus infection

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Laboratory methods using antibodies

Quantitation of Antigen by Immunoassays

Labeling and Detection of Antigens in Cells and Tissues

Measurement of Antigen-Antibody Interactions

Identification and Purification of Proteins

Agglutination(aggregation) Assays

Immunodiffusion

Complement Fixation

EIA enzyme immunoassay (IHCELISAELISPOT)

Immunofluorescence (IFA FACS)

ICS （ Intracellular cytokine staining ）

CLIA （ Chemiluminescence immumoassay ）

Traditional Immunoassays

Traditional Immunoassays

Antigen-Antibody Interactions

Modern Immunoassays

Modern Immunoassays

凝集反应 扩散试验 补体结合反应 凝集反应 扩散试验 补体结合反应

Immuno-labeling techniques免疫标记技术

a Principle

Specific Abs (or Ags ) labelled with fluorescein enzymes colloidial gold or radioisotopes are used as probes for the detection of Ags (or Abs)

b Types

IHC Western Blot fluorescence FACS microscope

CD4

CD8

Immuno-labeling techniques

horseradish peroxidase

EIA (Enzyme Immunoassay)

Substrate

Color Detection

Immuno-labeling techniques

IHC

Immunohistochemistry

免疫组织化学

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments They are typically labeled with probes that make them useful for detection purification or cell sorting applicationsSpecific secondary antibodies are selected according to the source of the primary antibody the class of the primary antibody (eg IgG or IgM) and the kind of label which is preferred

One conjugated 2nd antibody can detect all primary antibodies with same isotype which reduce the labor to conjugate all primary antibodies

Secondary antibody

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Objectives

1048698 To know the main laboratory detection method using antibody

To know how to isolate the specific lymphocyte population

1048698 Be able to describe how to detect the change of human immune function after bacteria or virus infection

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Laboratory methods using antibodies

Quantitation of Antigen by Immunoassays

Labeling and Detection of Antigens in Cells and Tissues

Measurement of Antigen-Antibody Interactions

Identification and Purification of Proteins

Agglutination(aggregation) Assays

Immunodiffusion

Complement Fixation

EIA enzyme immunoassay (IHCELISAELISPOT)

Immunofluorescence (IFA FACS)

ICS （ Intracellular cytokine staining ）

CLIA （ Chemiluminescence immumoassay ）

Traditional Immunoassays

Traditional Immunoassays

Antigen-Antibody Interactions

Modern Immunoassays

Modern Immunoassays

凝集反应 扩散试验 补体结合反应 凝集反应 扩散试验 补体结合反应

Immuno-labeling techniques免疫标记技术

a Principle

Specific Abs (or Ags ) labelled with fluorescein enzymes colloidial gold or radioisotopes are used as probes for the detection of Ags (or Abs)

b Types

IHC Western Blot fluorescence FACS microscope

CD4

CD8

Immuno-labeling techniques

horseradish peroxidase

EIA (Enzyme Immunoassay)

Substrate

Color Detection

Immuno-labeling techniques

IHC

Immunohistochemistry

免疫组织化学

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments They are typically labeled with probes that make them useful for detection purification or cell sorting applicationsSpecific secondary antibodies are selected according to the source of the primary antibody the class of the primary antibody (eg IgG or IgM) and the kind of label which is preferred

One conjugated 2nd antibody can detect all primary antibodies with same isotype which reduce the labor to conjugate all primary antibodies

Secondary antibody

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Laboratory methods using antibodies

Quantitation of Antigen by Immunoassays

Labeling and Detection of Antigens in Cells and Tissues

Measurement of Antigen-Antibody Interactions

Identification and Purification of Proteins

Agglutination(aggregation) Assays

Immunodiffusion

Complement Fixation

EIA enzyme immunoassay (IHCELISAELISPOT)

Immunofluorescence (IFA FACS)

ICS （ Intracellular cytokine staining ）

CLIA （ Chemiluminescence immumoassay ）

Traditional Immunoassays

Traditional Immunoassays

Antigen-Antibody Interactions

Modern Immunoassays

Modern Immunoassays

凝集反应 扩散试验 补体结合反应 凝集反应 扩散试验 补体结合反应

Immuno-labeling techniques免疫标记技术

a Principle

Specific Abs (or Ags ) labelled with fluorescein enzymes colloidial gold or radioisotopes are used as probes for the detection of Ags (or Abs)

b Types

IHC Western Blot fluorescence FACS microscope

CD4

CD8

Immuno-labeling techniques

horseradish peroxidase

EIA (Enzyme Immunoassay)

Substrate

Color Detection

Immuno-labeling techniques

IHC

Immunohistochemistry

免疫组织化学

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments They are typically labeled with probes that make them useful for detection purification or cell sorting applicationsSpecific secondary antibodies are selected according to the source of the primary antibody the class of the primary antibody (eg IgG or IgM) and the kind of label which is preferred

One conjugated 2nd antibody can detect all primary antibodies with same isotype which reduce the labor to conjugate all primary antibodies

Secondary antibody

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Laboratory methods using antibodies

Quantitation of Antigen by Immunoassays

Labeling and Detection of Antigens in Cells and Tissues

Measurement of Antigen-Antibody Interactions

Identification and Purification of Proteins

Agglutination(aggregation) Assays

Immunodiffusion

Complement Fixation

EIA enzyme immunoassay (IHCELISAELISPOT)

Immunofluorescence (IFA FACS)

ICS （ Intracellular cytokine staining ）

CLIA （ Chemiluminescence immumoassay ）

Traditional Immunoassays

Traditional Immunoassays

Antigen-Antibody Interactions

Modern Immunoassays

Modern Immunoassays

凝集反应 扩散试验 补体结合反应 凝集反应 扩散试验 补体结合反应

Immuno-labeling techniques免疫标记技术

a Principle

Specific Abs (or Ags ) labelled with fluorescein enzymes colloidial gold or radioisotopes are used as probes for the detection of Ags (or Abs)

b Types

IHC Western Blot fluorescence FACS microscope

CD4

CD8

Immuno-labeling techniques

horseradish peroxidase

EIA (Enzyme Immunoassay)

Substrate

Color Detection

Immuno-labeling techniques

IHC

Immunohistochemistry

免疫组织化学

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments They are typically labeled with probes that make them useful for detection purification or cell sorting applicationsSpecific secondary antibodies are selected according to the source of the primary antibody the class of the primary antibody (eg IgG or IgM) and the kind of label which is preferred

One conjugated 2nd antibody can detect all primary antibodies with same isotype which reduce the labor to conjugate all primary antibodies

Secondary antibody

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Laboratory methods using antibodies

Quantitation of Antigen by Immunoassays

Labeling and Detection of Antigens in Cells and Tissues

Measurement of Antigen-Antibody Interactions

Identification and Purification of Proteins

Agglutination(aggregation) Assays

Immunodiffusion

Complement Fixation

EIA enzyme immunoassay (IHCELISAELISPOT)

Immunofluorescence (IFA FACS)

ICS （ Intracellular cytokine staining ）

CLIA （ Chemiluminescence immumoassay ）

Traditional Immunoassays

Traditional Immunoassays

Antigen-Antibody Interactions

Modern Immunoassays

Modern Immunoassays

凝集反应 扩散试验 补体结合反应 凝集反应 扩散试验 补体结合反应

Immuno-labeling techniques免疫标记技术

a Principle

Specific Abs (or Ags ) labelled with fluorescein enzymes colloidial gold or radioisotopes are used as probes for the detection of Ags (or Abs)

b Types

IHC Western Blot fluorescence FACS microscope

CD4

CD8

Immuno-labeling techniques

horseradish peroxidase

EIA (Enzyme Immunoassay)

Substrate

Color Detection

Immuno-labeling techniques

IHC

Immunohistochemistry

免疫组织化学

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments They are typically labeled with probes that make them useful for detection purification or cell sorting applicationsSpecific secondary antibodies are selected according to the source of the primary antibody the class of the primary antibody (eg IgG or IgM) and the kind of label which is preferred

One conjugated 2nd antibody can detect all primary antibodies with same isotype which reduce the labor to conjugate all primary antibodies

Secondary antibody

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Agglutination(aggregation) Assays

Immunodiffusion

Complement Fixation

EIA enzyme immunoassay (IHCELISAELISPOT)

Immunofluorescence (IFA FACS)

ICS （ Intracellular cytokine staining ）

CLIA （ Chemiluminescence immumoassay ）

Traditional Immunoassays

Traditional Immunoassays

Antigen-Antibody Interactions

Modern Immunoassays

Modern Immunoassays

凝集反应 扩散试验 补体结合反应 凝集反应 扩散试验 补体结合反应

Immuno-labeling techniques免疫标记技术

a Principle

Specific Abs (or Ags ) labelled with fluorescein enzymes colloidial gold or radioisotopes are used as probes for the detection of Ags (or Abs)

b Types

IHC Western Blot fluorescence FACS microscope

CD4

CD8

Immuno-labeling techniques

horseradish peroxidase

EIA (Enzyme Immunoassay)

Substrate

Color Detection

Immuno-labeling techniques

IHC

Immunohistochemistry

免疫组织化学

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments They are typically labeled with probes that make them useful for detection purification or cell sorting applicationsSpecific secondary antibodies are selected according to the source of the primary antibody the class of the primary antibody (eg IgG or IgM) and the kind of label which is preferred

One conjugated 2nd antibody can detect all primary antibodies with same isotype which reduce the labor to conjugate all primary antibodies

Secondary antibody

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

凝集反应 扩散试验 补体结合反应 凝集反应 扩散试验 补体结合反应

Immuno-labeling techniques免疫标记技术

a Principle

Specific Abs (or Ags ) labelled with fluorescein enzymes colloidial gold or radioisotopes are used as probes for the detection of Ags (or Abs)

b Types

IHC Western Blot fluorescence FACS microscope

CD4

CD8

Immuno-labeling techniques

horseradish peroxidase

EIA (Enzyme Immunoassay)

Substrate

Color Detection

Immuno-labeling techniques

IHC

Immunohistochemistry

免疫组织化学

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments They are typically labeled with probes that make them useful for detection purification or cell sorting applicationsSpecific secondary antibodies are selected according to the source of the primary antibody the class of the primary antibody (eg IgG or IgM) and the kind of label which is preferred

One conjugated 2nd antibody can detect all primary antibodies with same isotype which reduce the labor to conjugate all primary antibodies

Secondary antibody

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Immuno-labeling techniques免疫标记技术

a Principle

Specific Abs (or Ags ) labelled with fluorescein enzymes colloidial gold or radioisotopes are used as probes for the detection of Ags (or Abs)

b Types

IHC Western Blot fluorescence FACS microscope

CD4

CD8

Immuno-labeling techniques

horseradish peroxidase

EIA (Enzyme Immunoassay)

Substrate

Color Detection

Immuno-labeling techniques

IHC

Immunohistochemistry

免疫组织化学

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments They are typically labeled with probes that make them useful for detection purification or cell sorting applicationsSpecific secondary antibodies are selected according to the source of the primary antibody the class of the primary antibody (eg IgG or IgM) and the kind of label which is preferred

One conjugated 2nd antibody can detect all primary antibodies with same isotype which reduce the labor to conjugate all primary antibodies

Secondary antibody

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

IHC Western Blot fluorescence FACS microscope

CD4

CD8

Immuno-labeling techniques

horseradish peroxidase

EIA (Enzyme Immunoassay)

Substrate

Color Detection

Immuno-labeling techniques

IHC

Immunohistochemistry

免疫组织化学

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments They are typically labeled with probes that make them useful for detection purification or cell sorting applicationsSpecific secondary antibodies are selected according to the source of the primary antibody the class of the primary antibody (eg IgG or IgM) and the kind of label which is preferred

One conjugated 2nd antibody can detect all primary antibodies with same isotype which reduce the labor to conjugate all primary antibodies

Secondary antibody

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

EIA (Enzyme Immunoassay)

Substrate

Color Detection

Immuno-labeling techniques

IHC

Immunohistochemistry

免疫组织化学

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments They are typically labeled with probes that make them useful for detection purification or cell sorting applicationsSpecific secondary antibodies are selected according to the source of the primary antibody the class of the primary antibody (eg IgG or IgM) and the kind of label which is preferred

One conjugated 2nd antibody can detect all primary antibodies with same isotype which reduce the labor to conjugate all primary antibodies

Secondary antibody

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

IHC

Immunohistochemistry

免疫组织化学

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments They are typically labeled with probes that make them useful for detection purification or cell sorting applicationsSpecific secondary antibodies are selected according to the source of the primary antibody the class of the primary antibody (eg IgG or IgM) and the kind of label which is preferred

One conjugated 2nd antibody can detect all primary antibodies with same isotype which reduce the labor to conjugate all primary antibodies

Secondary antibody

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

A secondary antibody is an antibody that binds to primary antibodies or antibody fragments They are typically labeled with probes that make them useful for detection purification or cell sorting applicationsSpecific secondary antibodies are selected according to the source of the primary antibody the class of the primary antibody (eg IgG or IgM) and the kind of label which is preferred

One conjugated 2nd antibody can detect all primary antibodies with same isotype which reduce the labor to conjugate all primary antibodies

Secondary antibody

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Secondary antibody

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Enzyme immunoassay (EIA)免疫酶测定法

EIA is to use enzyme-labeled Abs or Ags to detect Ag and Ab interactions

The enzyme converts a colorless substrate (chromogen) to a colored product

ELISA Ag or Ab in solution

Enzyme immunohistochemistry Ag in tissue

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

ELISA

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

ELISA

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Indirected ELISA

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

ELISA

BAS （ Biotin-avidin system ） -ELISA 生物素 - 亲和素放大系统 一分子亲和素可结合四个分子生物素敏感快速

生物素标记抗体亲和素标记酶

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Biotin-avidin system -ELISABiotin-avidin system -ELISA

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

灵敏度高单细胞水平可高通量筛选

（ Enzyme-linked immuno-sorbent spot ELISPOT)

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Two cytokines can be detected simultaneously

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Immunofluorescence 免疫荧光Immunofluorescence assay is to use a fluorescent compound (usually fluorescein) to detect the binding of Ag and Ab

The Ab is labeled with the fluorescent compound and its presence is revealed using a fluorescence microscope

Direct indirect immunofluorescence and indirect complement amplified immunofluorescence (间接免疫荧光互补放大 )

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

CD4

CD8

Immunofluorescence

Immunofluorescence ）Immunofluorescence ）

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

ImmunofluorescenceAn Example

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

(1) Useful diagram for fluorochomes properties

httpwwwbdbiosciencescomresearchmulticolorspectrumguideindexjsp

Useful tools

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

(2) Useful website tools

BD Fluorescence Spectrum Viewer httpwwwbdbiosciencescomresearchmulticolorspectrum_viewerindexjsp

Useful tools

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Identification and Purification of Proteins

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Immunoprecipitation A protein mixture is incubated with specifi c antibody Any antigenndashantibody complexes that form are precipitated from solution by the addition of Protein A-coated beadsthat bind to the antibodies and collect at the bottom of the tube under the force of centrifugation After washing the desired antigen is released from the antibody-boundbeads using altered pH andor high salt concentration

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Identification and Purification of Proteins

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Affinity ChromatographyAgarose beads bearing immobilized specifi c antibody are placed into a column with a semi-permeable plug at the bottom A solution containing antigen is passed slowly through the column allowing the binding of specifi c antigen to the immobilized antibody Unbound entities pass through the plug and any molecule that binds to the beads non-specifi cally is removed by extensive washing A solution with the appropriate pH and salt concentration to disrupt AgndashAb binding is then passed through the column to elute (wash off) the antigen of interest

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Western blotting

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Western Blot

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Western Blot

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Isolation of lymphocytes

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Magnetic cell sorting (MACS)

Three basic steps

1) Target cells are labeled with antibody-

conjugated magnetic particles

2) The labeled cells are placed within a magnetic field

3) The labeled cells are retained in the magnetic field while the unlabeled cells are washed away

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Cell subsets Purification

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

FACS separation( 流式细胞术分离 )

The basic principle of FACS is immunofluorescence and therefore flow cytometers can be considered to be specialized fluorescence microscopes

The modern flow cytometer consists of a light source collection optics electronics and a computer to translate signals to data

Isolation of different cell populations by FACS relies on the different expression of surface Ags

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Identification of cell subsets by FACS

Immune Cell types and subtypes defined by surface markers (CDs)

B cell

T cell

CD4+ T cell

CD8+ Tcells

Tregs (CD4+CD25+)

ConventionalCD4

Type of cell CD markers

stem cells CD34+CD31-

all leukocyte groups CD45+

Granulocyte CD45+CD15+

Monocytes CD45+CD14+

T lymphocyte CD45+CD3+

T helper cell CD45+CD3+CD4+

Cytotoxic T cell CD45+CD3+CD8+

B lymphocyte CD45+CD19+ or CD45+CD20+

Thrombocytes CD45+CD61+

Natural killer cell CD16+CD56+CD3-

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

bullFluidicsFluidics

bullCells in suspension

bullflow in single

bullOpticsOptics

bullscatter light and emit fluorescence

bullthat is collected filtered

bullElectronicsElectronics

bullconverted to digital values

bullStored amp analyzed on a computer

Collection

Drop charged and collected

Basics of Flow Cytometry

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Flow cytometry or Fluorescence Activated Cell Sorter (FACS)

Quantitative multi- parameter analysis of large numbers of individual cells

Cell surface markersIntracellular proteinsCa++ mobilization

12 colors and 15 parametersSorting 70000 cellsecond

FACS

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

FCM

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

T Lymphocyte Function Assays

Function Measurement --Proliferation-- CTL killing-- DTH--Apoptosis--Cytokine--HLA detection

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation

Morphology

3H-thymidine labeling

MTT

FACS-CFSE staining

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

T cell proliferation Morphology

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Lymphoblast(morphological

features)

Lymphoblasts are 12-20 microm in diameter with a round to oval nucleus The periphery of both the nucleus and the cell may be irregular in outline

The fine highly dispersed nuclear chromatin stains a light reddish-purple and one or two pale blue or colorless large nucleoli are visible The cytoplasm is usually basophilic with marginal (peripheral) intensity a common characteristic

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

T cell proliferation3H-thymidine labeling

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

T cell proliferation -MTT

Mitochondria enzyme catalyze the reduction of MTT ndash Turn blue

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

T cell proliferationFACS-CFSE staining

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

CTL Assay

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

1 Cytolysis (necrosis) ------ three stagesa contact phase recognition of antigen in the context of MHC class I moleculesb Polarization phase TCR co-stimulatory molecules cytolytic granules c lysis phase release perforin and granzymes osmotic death

CTL cytotoxicity mechanism

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

CTL Assay

Supernatant

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Control release ratio ( 》 5) average value

Control cpmndash Natural releaseMaximal release ndash Natural release

Specific release ratio( 》 10)Specific cpmndash Natural releaseMaximal release ndash Natural release

Natural release ratio( 《 15)

Controls Natural release well ndash medium Maximal release well ndash Detergent Lysed No antigen control

51Cr Release Assay

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

CTL ndash DIOCPI staining

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

MHC tetramers can be used to quantitate numbers of antigen-specific T cells (especially CD8+ T cells)

Biotinylated recombinant class I molecules folded with the peptide of interest and β2M and tetramerized by a fluorescently labeled streptavidin (Streptavidin binds to four biotins per molecule)

This tetramer reagent will specifically label T cells that express T cell receptors that are specific for a given peptide-MHC complex

Antigen specific responses can be measured as CD8+ tetramer+ T cells as a fraction of all CD8+ lymphocytes

Tetramer

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Tetramers can bind to three TCRs at once allowing specific binding in spite of the low (10-6 molar) affinity of the typical class I-peptide-TCR interaction

Tetramer

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

CD8 response related(now CD4 too) and known epitope

Infectious Disease

HIV-AIDS EBV CMV HPV HBV HCV Influenza Measles Malaria TB and RSV

Breast Prostate Melanoma Colon Lung Cervical Ovarian and Leukemia

Diabetes Lyme disease Multiple sclerosis Rheumatoid arthritis Autoimmune vitiligo

Transplantation

Autoimmune Disease

Tumor Immunology

Utility of Tetramer

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

DTH detection lsquoOTrsquo test or PPD test

结核杆菌素念珠菌素麻风菌素链激酶 - 链道酶腮腺炎病毒

Immunization

Challenge (EarFoogpad)

Measurement (Calipers)

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Apoptosis detection

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Apoptosis detection

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Flow cytometry of apoptotic cell death Phospholipid redistribution

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

In the TUNEL assayfragmented DNA is labeled by terminaldeoxynucleotidyl transferase (TdT)to reveal apoptotic cells When

Apoptosis detection

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

1Real-time PCR (mRNA Level)

2ELISAELISPOT

3Intracellular staining (FACS)

4 Biological Function

Cytokines detection

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Intracellular Staining

Identification of different T helper cells characterized by different cytokine production

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

血清学方法微量细胞毒试验或补体依赖的细胞毒试验（ CDC ）细胞学方法混合淋巴细胞反应分子生物学方法 PCR

HLA detection

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

HLA detection

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

HLA detection

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Assessment of human immune function

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Assessment of human immune function

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Content

Laboratory methods using antibodies

Lymphocytes isolation

Methods for studying lymphocyte

responses

Transgenic mice and gene targeting

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Transgenic animals and knockout animals

Part 1 Transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Transgenic AnimalAnimal has one or more foreign genes inserted into chromosome DNA inside its cells artificially

After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst foreign gene is inserted in a random fashion into chromosome DNA

Randomly (Foreign gene may disrupt an endogenous gene important for normal development and the chance is about 10 )multiple copies

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

ES cell transformation

Injection of gene into fertilized egg

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Method 1 ES cell transformation vs Method 2 Injection of gene into

fertilized egg

1 ES cell transformation works well in mice only Other transgenic animals are produced by egg injection

3 Injection of gene into fertilized egg is less reliable (viability of eggs frequency of integration)

but it helps to avoids chimeric animals

2 ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Applications of Transgenic Animals

Transgenic mice are often generated to 1 characterize the ability of a promoter to direct tissue-specific gene expression

eg a promoter can be attached to a reporter gene such as LacZ or GFP

2 examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals

3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse Intact animal provides a more complete and physiologically relevant picture of a transgenes function than in vitro testing

4 Drug testing

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Example 1 Transgenic MouseThe growth hormone gene has been engineered to be expressed

at high levels in animals

The result BIG ANIMALS

Metallothionein

promoter regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene Neural structures expressing the reporter transgene are dark blue-green

Example 2 Transgenic Mouse

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Tail tip95 day embryos -

GFP and wt

Example 3 GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

GFP transgenic mouse (Nagy)

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

knock-out AnimalOne endogenous gene in an animal is changed The gene can not be expressed and loses its functions

DNA is introduced first into embryonic stem (ES) cellsES cells that have undergone homologous recombination are identifiedES cells are injected into a 4 day old mouse embryo a blastocystKnockout animal is derived from the blastocyst

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animals

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

KNOCKOUT MICE

Isolate gene X and insert it into vector

Inactivate the gene by inserting a marker gene

that make cell resistant to antibiotic (eg Neomycin)

Transfer vector with (-) gene X

into ES cells (embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

eg(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombination

and gene disrution

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Generation of knockout mice

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells followed by +- selection

NeoR+ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element Recombinase would be expressed in accordance with specificity of your promoter

Promoter could be regulated artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life

Cre-lox technology

Cre ndash a site-specific recombinase enzyme from the P1 phage

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed)

If CRE recombinase expressed

Gene between loxP sites is removed

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Transgenic animals and knockout animals

Part 1 transgenic animalsIntroduction to transgenic animalsHow to make transgenic animalsApplications of transgenic animals

Part 2 Knockout animalsIntroduction to knockout animalsHow to make knockout animalsHow to make conditional knockout animalsApplications of knockout animal

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Applications of Knock-out animals

Find out if the gene is indispensable (suprisingly many are not)Check the phenotypes of knockout animalsDetermine the functions of knockout gene

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Three genome editing technologies

1 Zinc-finger endonucleases (ZFNs)

2 Transcription activatorndashlike effector nucleases (TALENs)

3 RNA-guided CRISPR-Cas9 nuclease system(CRISPR-Cas9)

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

2013 年十大科技突破

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

癌症免疫疗法大众基因显微手术脑成像技术人体胚胎克隆迷你器官宇宙射线的来源太阳能新星为什么睡觉微生物与健康疫苗设计

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

CRISPR-Cas9 system introduction

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

无论是 ZFN 还是 TALEN 这两种人工核酸酶的原理是一样的都是由 DNA 结合蛋白与核酸内切酶 Fok I 融合而成

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

CRISPRCas91987 年日本课题组在 K12 大肠杆菌的碱性磷酸酶基因附近发现串联间隔重复序列随后的研究发现这种间隔重复序列广泛存在于细菌和古细菌的基因组中2002 年科学家将其正式命名为 clustered regularly interspaced

short palindromic repeats(CRISPR) 2005 年三个研究小组均发现 CRISPR 的间隔序列 (spacer) 与宿主菌的染色体外的遗传物质高度同源推测其功能可能与细菌抵抗外源遗传物质入侵的免疫系统有关2007 年 Barrangou 等首次发现并证明细菌可能利用 CRSPR 系统对抵抗噬菌体入侵2008 年 Marraffini 等又发现细菌 CRISPR 系统能阻止外源质粒的转移首次利用实验验证了 CRISPR 系统的功能由此科学家们揭开了研究 CRISPR 系统作用机制的序幕

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Cas （ CRISPR associated） 存在于 CRISPR位点附近是一种双链 DNA核酸酶能在 guide RNA引导下对靶位点进行切割它与 folk酶功能类似但是它并不需要形成二聚体才能发挥作用

CRISPRCas 的基因座结构 基因座结构可分别三部分 5lsquo 端为 tracrRNA基因中间为一系列 Cas 蛋白编码基因包括Cas9 Cas1 Cas2 和 Csn2 3端为 CRISPR基因座由启动子区域和众多的间隔序列 (spacers)和重复序列(direct repeats)顺序排列组成

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Cas9 蛋白包含两个功能结构域一个在 N 端有类似于 Ruc 核酸酶的活性一个在中部有类似 HNH 核酸酶的活性

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

The crRNA and tracrRNA can be fused together to create a chimeric single-guide RNA (sgRNA)

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

利用 Cas9 系统建立 knock-out 细胞系

1 确定待敲除基因的靶位点

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

3 构建表达 sgRNA 的质粒

4 sgRNA 活性检测

5 利用 Cas9 系统建立 knock-out 细胞系

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

根据基因 ID 在 NCBI 或 ENSEMBLE 中进行查找 找到该基因 CDS 区分析相应的基因组结构明确 CDS 的外显子部分 对于蛋白编码基因如果该蛋白具重要结构功能域可考虑将基因敲除位点设计在编码该结构域的外显子如果不能确定基因产物性质可选择将待敲除位点放在起始密码子 ATG 后的外显子上

1 确定待敲除基因的靶位点

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

确定待敲除位点后选择 23- 至 250bp 的外显子序列输入到在线免费设计 sgRNA 的软件 Input 框中然后进行设计运算软件会自动输出 sgRNA 序列一般为 PAM 前的 20 个 bp 设计完成后要在 NCBI上比对一下确定没有其他互补基因 根据选择的 sgRNA 模板序列公司合成一对序列互补的 DNA Oligos

在线设计网址 httpcrisprmitedu

2 设计识别靶位点的识别的一对 DNA Oligo （引物）

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

将合成的 Oligos 以逐步降温的方法退火成双链然后与公司提供的质粒进行连接连接后转化感受态的大肠杆菌再进行涂板

挑单克隆培养提质粒酶切鉴定并且测序确认

从公司买 SgRNA 载体价格大概 2250 元 5T 3380 元 10T Cas9 质粒免费赠送

3 构建表达 sgRNA 的质粒

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

SSA sgRNA 活性检测试剂盒 1180 元 10T1980 元 20T

4 sgRNA 活性检测

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

将 Cas9 质粒和 SgRNA 转染细胞通过荧光标记观察转染效率如果选择了带 Neo 抗性的质粒则同时用 G418 药物筛选如果有 FACS 可直接分选带荧光的细胞 分离阳性细胞培养繁殖然后提取部分细胞的基因组 DNA 用作 PCR 模板然后用对应的引物进行 PCR 扩增 PCR 产物先进行 2琼脂糖凝胶电泳如果出现条带则将 PCR 产物直接送测序

将携带有突变等位基因的阳性克隆扩增保存稳转系建立成功

5 利用 Cas9 系统建立 knock-out 细胞系

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Cas9 sg-RNA

帅选阳性克隆构建成功

PAM 前 20bp

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

在人类基因组中平均每 127bp就出现一个 NGG PAM

Cas9 系统缺点 off target

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

2013 年 4 月 12日在《 cell stem cell》上发表的《 Enhanced Efficiency of Human Pluripotent Stem Cell

Genome Editing through Replacing TALENs with

CRISPRs》一文中作者利用 TALENs 和 CRISPRs对同一基因进行修饰效率分别为 0-34 和 51-79

CRISPR-Cas 系统前景分析

而且从实际应用的角度来说 CRISPRs 比 TALENs

更容易操作因为每一对 TALENs都需要重新合成而用于 CRISPR 的 gRNA只需要替换 20个核苷酸就行且Cas蛋白不具特异性

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html

Cas9 movies httpvyoukucomv_showid_XODIzNjAzNDI0html