Laboratory of Molecular Genetics, KNU Gene Cloning.

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

Gene Cloning


Cloning - a definition

• From the Greek - klon, a twig• An aggregate of the asexually produced

progeny of an individual;a group of replicas of all or part of a macromolecule (such as DNA or an antibody)

• An individual grown from a single somatic cell of its parent & genetically identical to it

• Clone: a collection of molecules or cells, all identical to an original molecule or cell


Joshua Ledergerg & Edward Tatum, 1946

두 종류의 다른 유전자를 가진 대장균 사이에 유전자의 재조합이 일어나 새로운 대장균이 만들어 지는 것을 발견했다 . 이 원리를 바탕으로 1970 년대에 DNA 재조합 기술이 발전하게 되었다 .

DNA technology (DNA DNA technology (DNA 조작기술조작기술 ))


1. 형질전환 (Transformation) 세포 주변에 있는 유전물질을 받아 들이는 방법 (Frederick Griffith 1920)

2. 형질도입 (Transduction) 세균의 유전자를 박테리오 파지가 전달

3. 접합 (Conjugaton)두 개의 세포사이에 접합을 통한 DNA 이동

원핵생물에서 원핵생물에서 DNADNA 의 이동방법 의 이동방법


The host cell : The host cell : Escherichia ColiEscherichia Coli

DNA 증식을 위해 transformation 중요Transformation 을 위해 요구되는 요소 - gene 도입을 위한 적당한 숙주 - 숙주 안으로 gene 을 도입할 운반체 - gene 을 받아들인 숙주 선별할 수단

Bacterium E. coli - 가장 넓게 사용 ; simple, genetic environment 잘 알려짐 - genome 완전히 분석됨 - genetic code 보편적이기 때문에 다른 생물의 외래 DNA 받아들임 → DNA 의 구성 , 구조 , 기본 mechanism 동일하므로 복제 가능 - 빠르게 분열하고 , cell 분열할 때마다 도입된 DNA 도 복제 - culture medium 에서 37℃ 일 때 최고 성장 - bacterial growth


0 ℃ 처리하여 세포막 고형화 ,

charged phosphate stabilizing

- Transformation solution 속의 양이온들이

phosphate group 과 complex 이룸

→ (-) charge 가리므로 DNA 분자 이동 가능

- Heat shock

→ 세포막의 열적 불균형 형성하여

adhesion zone 통한 DNA pumping 도움

Proposed molecular mechanism of

DNA transformation of E. coli


The Boyer-Cohen- Chang experiment. The Boyer-Cohen- Chang experiment. 19731973Proof that The Boyer-Cohen-Boyer-Cohen- Chang experiment created a recombinant DNA molecule


inserting new genes into plasmids - gene cloning technology ; cut and past DNA fragment - plasmid vector 는 cloning site( 제한효소 인식 서열 ) 포함 ; 원형의 plasmid 절단하여 open, DNA 삽입 가능 - 제한효소 → sticky end 형성 ; 보완적인 다른 fragment 와 수소 결합 형성하므로 DNA ligase 를 위해 충분한 시간 동안 DNA fragment 잡아둠 - DNA ligase ; 인접한 nucleotide 사이에 phosphodiester 결합 재형성 → stable double helix

DNA Recombination


Restriction endonucleasesRestriction endonucleases

cut DNA - DNA 의 특정 sequence 인지 , 절단 (break phosphodiester bond)

발견과정 -1950s; bacteria 에서 원시적인 immune system 발견 -1960s; enzyme system (E. coli 추출물 ) 발견 → self DNA 보호 , 외래 DNA 인지 - 절단 modification activity (methylation) -1970s; New restriction endonuclease 발견 → Hind (Ⅲ haemophilus influenzae 에서 발견 ) → modification activity 없음 , 인식자리 안의 정확한 지점 절단


Three major class - type , ; restriction and modification activity ,Ⅰ Ⅲ 인식자리 밖 절단 , ATP 를 에너지원으로 사용 → 예측 불가능 , ATP 요구성 때문에 사용 안 함 - type ; restriction activityⅡ 만 있음 , ATP 필요 없음 , Mg2+ 필요 , 인식자리 안이나 인접부위 예측 가능하게 절단 → DNA 조작에 이상적

절단방법

- middle of the site → blunt end - 3’ of center, 5’ of center → sticky endFrequency of cutting ; 제한효소가 인지하는 sequence 의 길이에 의존Restriction map ; 제한효소에 의해 절단한 DNA fragment 의 크기 비교 → genetic map 과 연관

Restriction endonucleasesRestriction endonucleases


Restriction enzymes cleave DNA at a specific sequence


Molecular detail of EcoR1 restriction-modification


HaeIII

Haemophilus aegiptius GG/CC Blunt cut

Sau3A

Staphylococcus aureus /GATC 5’-overhang

HhaI Haemophilus haemolyticus

GCG/C 3’-overhang

SmaI Serratia marcescens CCC / GGG Blunt cut

EcoRI Escherichia coli RY13 G / AATTC 5’-overhang

PstI Providencia Stuartii CTGCA / G 3’-overhang

HaeII Haemophilus aegiptius RGCGC / Y Ambiguous sequence

NotI Nocardia otitidis GC / GGCCGC

8 nt sequence

Properties of restriction enzymes-2


selectable markers - plasmid 삽입된 cell 과 삽입되지 않는 cell 구별을 위해 사용 - antibiotic resistance 이용 ; plasmid 삽입된 cell 만 항생제 함유 배지에서 생존 - antibiotic resistance → chemical modification 통해서 target antibiotics inactivation → 세포막을 통한 antibiotics transport 방해

Plasmid selection


플라스미드를 이용한 플라스미드를 이용한 cloning cloning

1. 미생물로부터 플라스미드를 분리한다 .

2. 동물 , 식물로부터 특정한 유전자가 포함된 DNA 를 분리한다 .

3. 분리한 유전자를 포함하고 있는 DNA 조각을 플라스미드에 삽입하여 재조합 DNA 를 만든다 .

4. 박테리아 세포에 재조합 플라스미드를 넣어 형질전환을 한다 .

5. 재조합 박테리아 클론이 확보 사용된다 .


TransformationTransformation

E. Coli

; CaCl2 + heat shock(42 ) ℃ 조건에서 형질전환 일어남

다른 이온 (Mg2+, Mn2+,Ba2+ 등 ) 도 사용 ; mixture of positive ion 사용시 → 효율 증가

DNA size 와 conformation 이 형질전환 효율에 영향A subset of cell 에 제한 받음 → plasmid 수 증가해도 형질 전환된 cell 수 변화 없음E. coli 가 DNA 받아들이는 정확한 메커니즘 밝혀지지 않음 → adhesion zone 가설 ; 세포막의 adhesion zone 에서 channel 형성단점 ; Large DNA 는 성공적으로 형질전환 되기 힘듦


Directional cloning


무차별 유전자 클로닝 방법

1. 제한효소를 이용하여 DNA를 수천 조각으로 절단

2. 각 DNA 조작은 서로 다른 벡터분자에 실려 박테리아 세포에 형질 전환

3. 수많은 종류의 박테리아 클론을 genomic library 라 함

Genomic library ( 유전자 도서관 )


The plasmid vectorpropagation of plasmids - bacterial cell 의 빠른 증식 능을 이용하여 특정 gene 을 증폭 시킬 때 plasmid 이용 (host cell division 시에 plasmid duplication) - origin of replication(ori) sequence 필요 → host cell 안에서 복제 가능하게 함 - 복제 조절에 따라 2 group 으로 구분 → strigent control ; bacterial cell 분열에 조절 받음 (1 개씩 replication) → relaxed ; bacterial cell 과 자율적 (cell 당 수 백개의 copy 축적 )


pBR322


pUC19


GST-Vector (pGEX 6p, T7)


Eukaryotic expression vector


Isolation of recombinant plasmidsIsolation of recombinant plasmids

1.E.coli 을 EDTA, Glucose 섞인 buffer로 현탁

2. SDS, NaOH Mixture 첨가 → cell lysis, DNA denature

3. potassium acetate, acetic acid 첨가 → neutralization

4. 상층액에 ethanol 이나 isopropanol 첨가 → plasmid DNA 침전

5. pellet = clean plasmid DNA

6. 전기 영동 하여 재조합 확인


DNA is negatively charged due to phosphates on its surface. As a result, it moves towards the positive pole.

Size separation of DNA fragments by electrophoresis in agarose gels


D0313

D0332

D0334

D0324

D0276

D0194

His Ku7

0

KLA6

KAP1FL

AG s

uv42

0DNM

T1

DNMT3

A2DNM

T3A34


It is a technique for correcting defective genes that are responsible for disease development

There are four approaches:1. A normal gene inserted to compensate for a nonfunctional gene.2. An abnormal gene traded for a normal gene3. An abnormal gene repaired through selective reverse mutation4. Change the regulation of gene pairs

Gene Therapy


How It Works

A vector delivers the therapeutic gene into a patient’s target cell

The target cells become infected with the viral vector

The vector’s genetic material is inserted into the target cell

Functional proteins are created from the therapeutic gene causing the cell to return to a normal state


The First Case

The first gene therapy was performed on September 14th, 1990 Ashanti DeSilva was treated for SCID

Sever combined immunodeficiency Doctors removed her white blood cells,

inserted the missing gene into the WBC, and then put them back into her blood stream.

This strengthened her immune system Only worked for a few months


http://encarta.msn.com/media_461561269/Gene_Therapy.html


Viruses

Replicate by inserting their DNA into a host cell

Gene therapy can use this to insert genes that encode for a desired protein to create the desired trait

Four different types


Retroviruses

Created double stranded DNA copies from RNA genome The retrovirus goes through reverse

transcription using reverse transcriptase and RNA

the double stranded viral genome integrates into the human genome using integrase integrase inserts the gene anywhere because

it has no specific site May cause insertional mutagenesis

One gene disrupts another gene’s code (disrupted cell division causes cancer from uncontrolled cell division)

vectors used are derived from the human immunodeficiency virus (HIV) and are being evaluated for safety


Adenoviruses

Are double stranded DNA genome that cause respiratory, intestinal, and eye infections in humans

The inserted DNA is not incorporate into genome

Not replicated though Has to be reinserted when more cells

divide Ex. Common cold


Adenovirus cont.

http://en.wikipedia.org/wiki/Gene_therapy


Adeno-associated Viruses

Adeno-associated Virus- small, single stranded DNA that insert genetic material at a specific point on chromosome 19

From parvovirus family- causes no known disease and doesn't trigger patient immune response.

Low information capacity gene is always "on" so the protein is always being

expressed, possibly even in instances when it isn't needed.

hemophilia treatments, for example, a gene-carrying vector could be injected into a muscle, prompting the muscle cells to produce Factor IX and thus prevent bleeding.

Study by Wilson and Kathy High (University of Pennsylvania), patients have not needed Factor IX injections for more than a year


Transgenic Mouse: Generic term for an engineered mouse that has a normal DNA sequence for a gene replaced by an engineered sequence or a sequence from another organism.

Knockout Mouse: A transgenic mouse in which the normal gene is missing or engineered so that is not transcribed or translated. “Knocks out” that gene.

Knockin Mouse: A transgenic mouse in which the engineered “transgene” is subtly manipulated to: (A) alter the function of the gene (e.g., replace one amino acid with another in a site to determine if that site is essential for the protein’s function); (B) change transcription rate to overproduce or underproduce the gene product; or (C) create a fluorescent gene product to map its distribution in tissue.

Conditional Knockout (Knockin) Mouse: A transgenic mouse in which the transgene is knocked out (or in) in specific tissues, at a specific developmental stage, or in response to an exogenous substance (e.g., an antibiotic).

Konckout Mice


Transgenic Organisms

General Outline:

• Infect blastocyst cells/sperm with viral vector with the gene of interest.

• Hope that in some cells homologous recombination will insert the DNA section of interest into the target cell’s chromosome.

•Select chimeric organisms.

•Breed until the transformed DNA is found in a germ line.


agcttatcgaat

cgatcgctag

Gene of Interest

UpstreamDNA (uniqueto the gene),usually > 1kb

DownstreamDNA (unique)to the gene,

usually > 1 kb

agcttatcgaat

cgatcgctag

Desired Gene Antibiotic Resistance Gene

(2) Construct the desired DNA sequence (i.e., the transgene), adding a gene for antibiotic resistance, but keeping the upstream and the downstream nucleotides.

(1) Get the nucleotide sequence of the gene of interest. Including upstreamand downstream nucleotides.


(3) Micropipette embryonic stem cells from the inner cell mass of ablastocyst (i.e. early mouse embryo) in a strain with a physically recognizablephenotype (e.g., pigmented).

(4) Culture the cells with many copies of the manufactured transgenicDNA complex. Short bursts of an electrical current allow the DNA to passthrough the plasma membrane into the cell (electroporation).


(5) Cells will divide in culture and some of them will incorporate the transgenic DNAstrand into the chromosome (homologous recombination). After a sufficient numberof cell divisions, add the antibiotic. This will preferentially kill those stem cells thathave not incorporated the transgenic strand (black dots), giving a good harvest ofthose that have incorporated the strand (red dots).

+ antibiotic

(6) Insert the stem cells into the blatocyst of a mouse with a different geneticbackground trait (e.g., an albino if the original stem cells came from a pigmented mouse).


(7) Implant the new blastocysts into a pseudopregnant female with a visiblephenotype different from the blastocyst phenotype (e.g., albino if the blastocyst is pigmented).

(8) Offspring that have pigmented sections are chimeras that have incorporatedthe transgenic sequence into their cell lines. Select them for further breeding.


(9) Keep breeding the offspring of the chimeras until some fully pigmented miceare born. A fully pigmented mouse means that the transgenic germline generatedone of the gametes that resulted in that mouse. Genotype the mouse to determinethe genotype at the desired locus and the insertion point(s). (Most will be heterozygotes for the wild type allele and the transgenic allele).

(10) Mate two heterozygotes and genotype their offspring. This will give all threegenotypes--wild type homozygotes, heterozygotes, and transgenic homozygotes.

(11) Compare the three genotypes on the phenotype of interest.

Laboratory of Molecular Genetics, KNU Gene Cloning.

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