Laboratory of Molecular Genetics, KNU Gene expression & Analysis.

Laboratory of Molecular Genetics, KNULaboratory of Molecular Genetics, KNU

Gene expression & Analysis


Where are the genes located?Where are the genes located?

Genes are located Genes are located on the on the chromosomes.chromosomes.

Every species has Every species has a different number a different number of chromosomes.of chromosomes.

There are two There are two types of types of chromosomes: chromosomes: autosomes and sex autosomes and sex chromosomeschromosomes


• Genes are located on the chromosomes which are found in the nucleus of a cell.

• When a cell is undergoing cell reproduction, the chromosomes are visible. Chromosomes appear when the chromatin condenses and become visible.

• Most of the time (90%) the genetic material in the form of chromatin.

• A genome is the complete genetic information contained in an individual.– (gene + chromosome)


What is gene expression?What is gene expression?

Gene expression is Gene expression is the activation of the activation of a genea gene that that results in results in a a proteinprotein..


Gene expression takes place differently in Gene expression takes place differently in prokaryotes and eukaryotes. prokaryotes and eukaryotes.

ProkaryotesProkaryotes– No membrane bound No membrane bound

organelles (nucleus)organelles (nucleus)– More primitive More primitive

organismsorganisms– Only one circular Only one circular

chromosomechromosome– Bacteria are the only Bacteria are the only

organisms that are organisms that are prokaryotes.prokaryotes.

EukaryotesEukaryotes– Membrane bound Membrane bound

organelles ( specialize in organelles ( specialize in function –nucleus, function –nucleus, mitochondria, chloroplast)mitochondria, chloroplast)

– Chromosomes are in pairs Chromosomes are in pairs and not circularand not circular

– All organisms that are not All organisms that are not bacteria: protist, fungi, bacteria: protist, fungi, plants and animalsplants and animals


DNA in eukaryotes has regions of coding DNA in eukaryotes has regions of coding and noncoding DNA. The regions of DNA and noncoding DNA. The regions of DNA that code for proteins or traits are called that code for proteins or traits are called EXONSEXONS, while the regions that do not , while the regions that do not code for proteins are called code for proteins are called INTRONS.INTRONS.

cytoplasm


In Eukaryotes, following mitosis or meiosis, In Eukaryotes, following mitosis or meiosis, DNA recoils but certain regions remain relaxed DNA recoils but certain regions remain relaxed for transcription. The areas of relaxed DNA for transcription. The areas of relaxed DNA are called are called euchromatineuchromatin..

Transcription is theTranscription is the

Reading of the DNA andReading of the DNA and

Changing the code toChanging the code to

mRNA.mRNA. Translation is changingTranslation is changing

The mRNA into a trait byThe mRNA into a trait by

Using tRNA to interpret theUsing tRNA to interpret the

mRNA.mRNA.


RNARNA– Single strandedSingle stranded– Does not contain Does not contain

thymine but has uracil thymine but has uracil instead.instead.

tRNA carries 3 base tRNA carries 3 base pair code for specific pair code for specific amino acid.amino acid.

Amino acids compose Amino acids compose polypeptid chains.polypeptid chains.

One or more One or more polypeptide chains polypeptide chains compose a proteincompose a protein

proteins provide the proteins provide the “blueprints” for our “blueprints” for our characteristics and characteristics and functions.functions.

Translation


Southern hybridization

핵산분리

제한효소처리

Agarose 전기영동

Southern blot

Gel 전처리

Hybridization & detection


Southern(1975) 에 의해 개발된 방법으로 , 그 순서를 요약하면 다음과 같다 . - 해석할 DNA 를 적당한 제한효소로 절단한다 . - 절단된 DNA 를 agarose gel 전기영동에 의해 분자량의 순으로 분획한다 . - 변성된 DNA 를 agarose gel로부터 nitrocellulose filter 에 흡착시켜 고정시킨다 .- 이렇게 준비된 filter 와 방사선 동위원소로 표지한 probe 를 수용액 중에서 hybridization - Filter 를 X 선 필름에 노출시켜 방사선으로 감광된 band 를 검출함으로써 목적하는 DNA 의 존재를 확인하는 것이다 .

Southern Blotting 원리


Southern Blotting 실험방법

1. DNA 의 절단목적의 DNA 를 적당한 제한효소로 절단한다 .2. Agarose gel 전기영동Agarose gel 은 두께 5 ~ 8mm 정도가 적당하며 , 크기는 DNA 분석용 gel 보다 큰 것이 좋다 . Agarose gel 전기영동은 보통 사용하는 방법과 같다 .3. DNA 단편을 Agarose gel 로부터 filter 에 흡착DNA 단편들을 Agarose gel 로부터 filter 에 흡착하는 방법으로는 1) capillary transfer2) Electrophoretic transfer3) Vacuum transfer 등의 3 가지가 있다 . 여기에서는 가장 널리 쓰이는 capillary transfer 에 대하여 설명한다 . gel 이 두꺼운 경우 , 용액이 gel 로 침루하는 시간이 다르기 때문에 두께 5 ~ 8mm 정도의 gel 을 기준으로 한다 . 4. Hybridization hybridization 에 영향을 미치는 것은 , 오노 , 염농도 , 시간 , probe 의 농도 등이다 . 그러 나 특별한 경우를 제외하고는 온도는 65°C, 0.5 ~ 1.0M Nacl 농도에서 20 시간 정도로 한다 . Hybridization 방법은 , 사용하는 probe 의 종류 (DNA, 합성 oligonucleotide, RNA) 따라 다르다 , 그러므로 여기서는 가장 기본이 되는 DNA probe 를 사용한 경우를 설 명하고자 한다 .(1) 건조된 filter 를 내열성 비닐포에 넣고 10mL 의 prehybridization 용액을 가하여 적신 후 , 기포를 제거하고 끝부분은 접착기로 봉한다 .(2) 65°C, water bath 에서 2 ~ 3 시간 incubation 한 후 비닐포의 한쪽 끝을 약간 절단하여 prehybridization 용액을 제거한다 .(3) 5mL 정도의 hybridization 용액 및 probe(25¡ 50ng) 을 넣고 , 기포를 제거한 후 다 시 비닐포의 한쪽 끝을 봉하여 , 65 ~ 68°C 에서 16 ~ 24 시간 incubation 한다 .(4) Hybridization 이 끝난 filter 를 적당한 용기에 넣어 , 2X SSC-0.1% SDS 로 65 ~ 68 에서 10 분간 천천히 흔들어 주면서 2 회 세척한다 .(5) 다시 1X SSC-0.1% SDS, 0.3X SSC-0.1% SDS, 0.1X SSC-0.1% SDS 용액 순서 대로 65 ~ 68°C, 20 분간 천천히 흔들어 주면서 세척한 후 , Whatman 3 MM paper 위에 올려놓고 공기 중에서 건조시킨다 .(6) Filter 를 두장의 비닐랩 사이에 놓은후 , 이것을 X-ray exposure cassette 안에 놓 고 , 접착 테이프를 이용하여 고정 시킨다 .5. Autoradiography 및 X-ray flim 현상다음 (1), (3) 단계는 암실 , 안전등 (RED FILTER) 하에 실시한다 .(1) 고정된 filter 위에 X-ray film 를 올려놓아 고정시킨후 , 그 위에 intensifying screen 을 올려놓고 cassette 뚜껑을 덮는다 .(2) 실온 또는 -70°C 에서 2 ~ 3 시간 , 경우에 따라서는 1 ~ 2 일 노출시킨다 .(3) Film 을 film holder 에 걸어서 Kodak X-ray 현상액에서 2 ~ 3 분 정도 담근다 .(4) Film 을 흐르는 물속에서 4 ~ 5 분간 세척한다 .(5) Kodak fixer 액에 2 ~ 3 분간 담근다 .(6) Film 을 흐르는 물속에서 10 ~ 30 분 동안 세척한다 .(7) Film 을 걸어서 말린 후 , 확인한다 .


RNA 분리

1) Add 1 ㎖ TRIZOL 2) 5 min incubation at 15-30℃3) add 200 ㎕ chloroform. Shake tube vigorously by hand for 15 sec.4) 2-3 min incubation at 15-30℃5) Centrifuge at 13,200×rpm for 15 min at 2-8℃6) Transfer upper aqueous phase7) Add 500 ㎕ isopropyl alcohol (Precipitate the RNA) mix.8) Incubate sample at 15-30 for 10 min℃9) Centrifuge at 13,200×rpm for 10 min at 2-8 → pellet ℃ 없을 땐 1 번 더 spin.10) Remove supernatant( 빠르게 제거 )11) Wash the RNA pellet once with 75% EtOH 1 ㎖ (store 가능 : -20 )℃12) Mix the sample by vortexing and Centrifuge at 13,200×rpm 5min at 2-8℃13) Dry(air dry for 5min)14) Add DEPC H2O 25 ㎕


기본적으로는 Southern blotting 과 동일하다 . RNA 를 전기 영동한 후 filter 상에 고정하여 DNA( 또는 RNA) 를 probe 로 하여 hybridization 해서 목적의 RNA 를 검출한다 . 그러나 RNA 는 이차구조를 만들기 쉽기 때문에 gel 중에서 변성된 상태로 전기 영동을 시행한다 . (1) 취급에 따른 일반적 주의 RNA 에서 바른 정보를 얻기 위해서는 RNA 를 가능한 한 분해되지 않은 상태로 조제하지 않으면 안된다 . 특히 RNase 에 의한 분해에 주의할 필요가 있다 . RNase 의 오염으로부터 시료를 지키기 위하여 고무장갑 , disposable 제품 , Autoclave, 건열 멸균 , RNase 저해제 등을 이용한다 . 유리기구 , teflon 제 기구는 세정후 증류수로 잘 헹구어서 건열한다 . 전기 영동조 등 건열할 수 없는 기구는 세정 후 5% 의 과산화수소수에 1 시간 담그고 증류수로 잘 헹군다 . 물은 Autoclave 하여 사용한다 . 시약은 autoclave 하든가 할 수 없는 것은 여과 멸균한다 . 또 RNA 는 DNA 와 달리 alkali 에서 가수 분해되기 쉽다 . 완충액 등의 pH 가 alkali 로 되지 않도록 주의 할 필요가 있다 . (2 ) 추출조제 total RNA 나 poly(A)+RNA 를 이용한다 . RNA 를 조제한 경우 , 그 중 80% 정도가 rRNA 이며 mRNA 중의 정보를 얻기 위해서는 가능한 한 rRNA 의 혼입을 방지하는 것이 좋다 . 목적의 mRNA 가 적다고 생각되는 경우는 RNA 를 Oligo(dT) column 처리를 한다 . mRNA 는 poly(A) 구조를 3` 말단에 가지고 있기 때문에 이 조작으로 mRNA 를 수 십배로 농축 정제가 가능하다 . 그 외 단백질 혼입도 가능한 한피하지 않으면 안된다 .

1975 년 Southern EM 이 제한효소를 절단해서 agarose gel 전기 영동으로 분획한 DNA 단편을 직접 Nitro-cellulose filter 에 옮기는 방법을 개발했다 . 이 Southern blot 는 유전자 해석 수단으로서 아주 유용하기 때문에 그 후 널리 이용되고 있다 . 1977 년 Stark 는 agarose gel 전기 영동으로 분획한 RNA 를 filter 에 옮기는 방법을 개발했다 . 이 방법은 DNA 에 대한 Southern blot 와 대비시켜 Northern blot 로 부르게 되었다 .

Northern Blotting 원리


1. agarose gel 의 제작 (1% agarose, 1 x MOPS, 2.2mol/l formaldehyde)

2. RNA sample 의 조제 (RT-PCR RNA 준비과정과 동일 ) 3. 전기영동 (UV 조사 ) - 100 - 120V, 2 - 3 시간 (2 - 3 분간 ) 4. gel 을 꺼내고 10 x SSC 에 담근다 - 20 분 - 1시간 (NaCl, sodium citrate) 5. transfer - 하룻밤 overnight 6. 건열 처리 - 80 , 30℃ 분 - 2 시간 7. prehybridization - 42 , 2 - 3℃ 시간 8. hybridization - 42 , ℃ 하룻밤 overnight 9. 세 정 - 1 x SSC, 0.1% SDS, 실온 , 20 분간 1 회 0.2 x SSC, 0.1% SDS, 68 , 20℃ 분간 3 회 10. autoradiography

Northern Blotting 실험방법


Question: You are using Northern blotting to analyze two mRNA samples derived from fibroblasts and hepatocytes. What will you see if you use a probe made from exon EIIIB of the fibronectin gene? What about using a probe made from the exon next to EIIIB?

Detection of alternative splicing by Northern blotting•Northern blotting can be used to detect specific RNAs in complex mixtures.•Southern blotting detects specific DNA fragments.•Western blotting (immunoblotting) detects specific proteins with antibodies.

RNA

RNAmixture

Transfer solution


■ RT-PCR 은 세가지 과정

(1) RNA 분리 과정 ( 이 과정은 Northern Blot 을 하기 전에 시행해야 하는 동일한 과정이다 ) (2) cDNA 합성 과정 (reverse transcription) (3) PCR amplification ( 이 과정은 Genomic DNA 로부터 특정 유전자 부위를 증폭시키는 과정과 같다 )

위의 세가지 과정으로 진행된다 . mRNA 로부터 reverse transcriptase 를 이용하여 cDNA 를 제조하는 방법에는 어떤 oligonucleotide 를 primer 로 사용하는가에 따라 세가지 방법 즉 ,(1) Antisense primer(3' 쪽 유전자에 특이성을 지닌 primer) 를 이용하여 특정 부위 cDNA 제조(2) Random hexamer 를 이용하여 전체 mRNA 에 상보적인 cDNA 제조(3) Oligo dT primer 를 이용하여 전체 mRNA 에 상보적인 cDNA 제조가 있다 . 여기서는 oligo dT primer 를 이용한 방법을 사용하였다 .또한 Northern Blot 과 마찬가지로 cDNA 가 만들어질 때까지는 RNase 에 의한 오염 방지에 신경을 써야 한다 .

RT-PCR 과정


RT-PCR 원리

RT-PCR 이란 P.Seeburg(1986) 에 의해 RNA 를 찾고 분석하는데 도입된 방법으로 mRNA(messenger RNA)로부터 reverse transcription 과정을 통해 얻어진 cDNA(complementary DNA) 를 PCR 로 증폭하는 방법이다 . 이러한 방법은 RNA 검사의 sensitivity 를 높이고 소량의 RNA 로부터 염기서열을 분석할 수 있게 하였다 .

맨 먼저 reverse transcriptase 로 RNA 를 complementary DNA(cDNA) 로 역전사합니다 . 이때 쓰이는 primer 는 downstream primer뿐입니다 . 왜냐하면 RNA 는 single strand 이기 때문입니다 . 그후 생성된 cDNA 에서 일반적인 PCR 의 과정을 밟아서 유전자를 증폭합니다 .


그러면 downstream primer 는 어떻게 만들까요 . 크게 다음과 같은 세가지 방법이 알려져 있습니다 . mRNA 뒤에 poly(A) tail 이 붙는 것을 응용한 oligo(dT) primer, gene specific primer, 그리고 여기저기 붙는 random primer 입니다 .

Oligo(dT) primer 를 쓰면 분리한 RNA 중 mRNA 는 모두 cDNA 로 만들어집니다 . Gene specific primter 를 사용하면 target gene 만 cDNA 로 만들어지겠지요 . 그리고 random primer( 주로 hexamer) 는 염기서열이 일정치 않지만 길이가 고정된 primer 입니다 . 이런 primer 를 넣고 반응시키면 길이도 다양하고 모든 RNA(rRNA, tRNA 포함 ) 가 모두 cDNA 로 만들어지겠지요 .


DNA synthesis is done by an enzyme (DNA polymerase) adding nucleotides to the 3’-

end of a primer DNA chain


Polymerase Chain Reaction (PCR)-1A pre-defined DNA

sequence in the genome can be greatly amplified by repeated Polymerization cycles using 2 primers which hybridize to the ends of the target DNA. In

each cycle, the amount of target DNA

is doubled. After 10, 20 and 30 cycles, there is a

1000-, million- and billion-fold amplification

respectively.


Polymerase Chain Reaction (PCR)-2

Each PCR cycle has 3 steps-

a. Melting of DNA

b. Hybridization of primer

c. DNA synthesis


Western blot

• Antigen- Antibody reaction 을 이용하여 여러 단백질 혼합물 중에서 원하는 특정 단백질만을 찾아내는 방법

Block solution

Nitrocellulose membrane

antigen

Primary antibody

Secondary

antibody


Western Blotting 원리


•Polyacrylamide Gel Electrophoresis:– Break protein complexes into individual proteins– Separates protein samples based on size

•Western Blot Analysis:– Transfer the proteins to a nitrocellulose membrane– More stable and permanent– Identifies proteins by immunodetection: using

specific antibodies against the protein of interest

From Gel to Blot


Mini Trans-Blot Transfer Cell


Prepare for Electrophoretic Transfer

• Place the closed and locked cassette in the electrode module

• Add the frozen Bio-Ice cooling unit and place in tank

• Fill the tank with buffer

• A stir bar can be added to help maintain the ion and temperature distribution in the tank even


Transfer Proteins from the gel to the nitrocellulose membrane

30 minutes100V

Blotting buffer 1x Tris glycine with 20% ethanol

Ele

ctri

c C

urr

en

t


Add the Primary Antibody

anti-myosin light-chain

Wash

•Discard blocking solution

•Pour 10ml of primary antibody onto the membrane and gently rock for 10 minutes

•Primary antibody will bind to the myosin light-chain

•Quickly rinse membrane in 50ml of wash buffer and discard the wash buffer

•Add 50ml of wash leave for 3 minutes on the rocking platform


Add Enzyme-linked Secondary Antibody

Wash

•Discard wash solution

•Pour 10ml of the secondary antibody onto the membrane and gently rock for 10 minutes

•Secondary antibody will bind to the primary antibody

•Quickly rinse membrane in 50ml of wash buffer and discard the wash buffer

•Add 50ml of wash leave for 3 minutes on the rocking platform

Western Blot animation


A. SAMPLE PREPARATION

Sample preparation procedures are provided for monolayer cells, suspension cells, and tissue samples. Follow the procedure suited to your needs.

Monolayer Cells

• Grow cells to subconfluency in a 100 mm x 20 mm petri dish, remove culture medium and rinse cell monolayer with room temperature 1x PBS. The following steps should be performed on ice or at 4° C using fresh, ice cold buffers.

• Add 0.6 ml of RIPA buffer to the monolayer cells in the plate. Gently rock plate for 15 minutes at 4° C. Remove adherent cells with a cell scraper. Transfer the resulting lysate to a microcentrifuge tube.

• Wash the plate once with 0.3 ml of RIPA buffer and combine with first lysate. (Optional: Add 10 µl of 10 mg/ml PMSF and/or pass through a 21-gauge needle to shear the DNA.) Incubate 30–60 minutes on ice.

• Centrifuge cell lysate at 10,000xg for 10 minutes at 4° C. The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microcentrifuge tube. This is your whole cell lysate. For increased protein recovery, resuspend the pellet in a small volume of RIPA, centrifuge and combine supernantants.

Suspension Cells

• Collect approximately 2.0 x 107 cells by low-speed centrifugation (e.g. 200xg) at room temperature for 5 minutes. Carefully remove culture medium.

• Wash the pellet with PBS at room temperature, and again collect by low-speed centrifugation. Carefully remove supernatant.

• Add 1.0 ml of ice cold RIPA buffer with freshly added (Protease Inhibitors) and/or (Phosphatase Inhibitors). Gently resuspend cells in RIPA buffer with a pipet and incubate on ice for 30 minutes.

• Further disrupt and homogenize cells by hydrodynamic shearing (21-gauge needle), dounce homogenization or sonication, taking care not to raise the temperature of the lysate. (Optional: Add 10 µl of 10 mg/ml PMSF) Incubate 30 minutes on ice.

• Transfer to microcentrifuge tube(s) and centrifuge at 10,000xg for 10 minutes at 4° C. The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube. This is your whole cell lysate. For increased protein recovery, resuspend the pellet in a small volume of RIPA, centrifuge and combine supernantants.

Western Blotting 실험과정


B. ELECTROPHORESIS

• Mix sample (40–60 µg whole cell lysate, 10–20 µg nuclear extract or 10–20 ng purified protein per lane) with an equal volume of 2x electrophoresis sample buffer and boil for 2–3 minutes. Unused samples may be stored at -20° C.

• Load up to 10 µl of lysate per 1.0 mm of well width for gels of 0.75 mm thickness.

• We recommend the use of Cruz Marker™ molecular weight standards. Load 2 µl/well for 0.75 mm gels and 5 µl/well for 1.5 mm gels. When used with Cruz Marker™ compatible secondary antibodies, internal standard bands will appear when the probed blot is exposed to detection reagent. Alternatively, use Prestained Molecular Weight Standards.

• Electrophorese according to standard protocols.

• Transfer proteins from the gel to a nitrocellulose or PVDF membrane using an electroblotting apparatus according to the manufacturer´s protocols.

C. IMMUNOBLOTTING

• Block non-specific binding by incubating membrane in Blotto (either Blotto A or Blotto B; IgG-free BSA, is recommended when using anti-bovine secondary antibodies) for 30–60 minutes at room temperature. Alternatively, the membrane may be blocked at 4° C overnight in a covered container, using Blotto without Tween-20.

• If using a phospho-specific antibody, add 0.01% (v/v) of each Phosphatase Inhibitor Cocktails to the blocking solution and the antibody diluent to inhibit phosphatases.

• Incubate the blocked membrane in primary antibody diluted in Blotto for 1 hour at room temperature. (For phospho-specific antibodies: Use Blotto B with 0.01% (v/v) of each Phosphatase Inhibitor Cocktails Optimal antibody concentration should be determined by titration. We recommend a starting dilution of 0.5-2.0 µg/ml. Wash membrane three times for 5 minutes each with TBST.

• Incubate the membrane for 45 minutes at room temperature with horseradish peroxidase (HRP) conjugated secondary antibody, or alkaline phosphatase (AP) conjugated secondary antibody (Conventional Secondary Antibodies for Western Blotting), diluted to 1:500–1:2000 in Blotto. If high backgrounds are observed, secondary antibody should be diluted further (up to 1:20,000). If Cruz Marker™ molecular weight standards are used in the gel, the Cruz Marker™ compatible secondary antibodies must be used in order to visualize standards with ECL.

• Wash membrane three times for 5 minutes each with TBST and once for 5 minutes with TBST.

• Incubate membrane in Chemiluminescence Luminol Reagent according to Luminol data sheet, or visualize proteins using standard protocols. If luminol is used for visualization, an HRP-conjugated secondary antibody must be used.


Transfection and Protein localization


Exploring protein function1) Where is it localized in the cell?

2) What is it doing in the cell?

Approaches:a) Make antibodies - immunofluorescence

Approaches:a) Reduce protein levels - RNA

interference

b) “Express” the protein in cells with a tag Fuse to GFP

b) Increase protein levels “over-express”

c) “Express” mutant versions

Transfection!!!!


Transfection = Introduction of DNA into mammalian cells

Gene is transcribed and translated into protein= “expressed”


Direct introduction of the DNA

Electroporation - electric field temporarily disrupts plasma membrane

Biolistics (gene gun)- fire DNA coated particles into cell

Microinjection


Infection:Use recombinant viruses to deliver DNA

RetrovirusesAdenoviruses

Virally-mediated introduction of the DNA


Positively charged carrier molecules are mixed with the DNA and added to cell culture media:

Calcium PhosphateDEAE Dextranliposomesmicelles

Carrier-DNA complexes bind to plasma membrane and are taken up

Carrier-mediated introduction of the DNA


Types of Transfection

Transient:Expression assayed 24-48

hours post transfection

Stable:Integration of the transfected

DNA into the cell genome - selectable marker like neomycin resistance required

“stably transfected” cell line


DNA “expression” vector transfected:

pCMV/GFP

CMV

Promote

r

Insert genein here

Polyadenylationsite

SV40

Pro

mote

rN

eom

ycin

resista

nce

PolyadenylationsitepUC

Bacterial origin of replicationA

mpicillin

resista

nce

For expression in cells

To generate stable cell line

For amplification of the plasmid in bacteria

GFP


PROTEIN X

PROTEIN Y

GFP

GFP

GFP ZPROTEIN

Three ways to make Green fluorescent protein “GFP” fusion constructs:


EXPERIMENT:

Transfect unknown GFP fusion proteinProtein X, Y or Z

Visualize GFP protein fluorescence by fluorescence microscopy in living cells

Counter-stain with known marker to compare localization patterns in living cells

= “vital stain”


Transfection 원리


Procedure1. The day before transfection, seed 2–8 x 105 cells per 60 mm dish in 5 ml appropriate growth medium. The cell number seeded should produce 40–80% confluence on the day of transfection.2. Incubate the cells under their normal growth conditions (generally 37°C and 5% CO2).3. On the day of transfection, dilute 5 µg DNA with cell growth medium containing no serum, proteins, or antibiotics to a total volume of 150 µl. (For primary cells, use 2.5 µg plasmid DNA). Mix and spin down the solution for a few seconds to remove drops from the top of the tube.4. Add 30 µl SuperFect Transfection Reagent to the DNA solution. (For primary cells, use 15 µl SuperFect Reagent). Mix by pipetting up and down 5 times, or by vortexing for 10 s.5. Incubate the samples for 5–10 min at room temperature (15–25°C) to allow transfection-complex formation.6. While complex formation takes place, gently aspirate the growth medium from the dish, and wash cells once with 4 ml PBS (phosphate buffer). 7. Add 1 ml cell growth medium (containing serum and antibiotics) to the reaction tube containing the transfection complexes. Mix by pipetting up and down twice, and immediately transfer the total volume to the cells in the 60 mm dishes.8. Incubate cells with the transfection complexes for 2–3 h under their normal growth conditions.9. Remove medium containing the remaining complexes from the cells by gentle aspiration, and wash cells once with 4 ml PBS.10. Add fresh cell growth medium (containing serum and antibiotics). Assay cells for expression of the transfected gene after an appropriate incubation time.

Transfection 실험방법


Transformation 과 Transfection- 제일 큰 차이는 host cell 이다 . Transformation 같은 경우는 박테리아 셀 내에 외래 DNA 를 주입하는 것이고 , transfection은 동물 세포에 주입하는 것을 말한다 . 동물세포에는 transformation 이 아닌 transfection 이란 용어를 사용하는 이유는 동물세포에서의 transformation 은 세포를 continuous cell line 로 변형시킨다는 의미를 이미 가지고 있기 때문이며 , 처음에 동물세포에 이뤄진 외래 DNA 주입 실험이 phage DNA 를 벡터로 사용해 이뤄졌기 때문에 바이러스 infection에서 어미를 따서 transfection 이란 용어를 사용하게 되었다 .

Transformation 과 Transfection

Competent cell 의 제조방법

LB plate 에 자란 DH5 alpha colony 를 LB 배지 2 ml 에 접종한 후 밤새 키웁니다 . 위에서 키운 배양액 1 ml 을 100 ml LB 배지가 담긴 플라스크에 넣고 O.D.600 이 0.4 가 될 때까지 37°C shaking incubator 에서 키웁니다 . 배양액을 50 ml conical tube 에 넣고 얼음에 10 분간 놓아둡니다 . 4°C 에서 4,500 rpm 으로 10 분간 원심분리합니다 . 상층액을 완전히 제거한 후 미리 차게 해 둔 0.1 M filtered CaCl2 용액을 원래의 1/2 부피로 넣고 부유시킵니다 . 얼음에 15 분 놓아두었다가 다시 4°C 에서 3,000 4,000 rpm∼으로 10 분간 원심분리합니다 . 상층액을 버리고 처음의 1/10 부피의 filtered CaCl2 용액으로 부유시킵니다 . 얼음에 30 분 놓아두었다가 미리 차게 해둔 microfuge tube 에 200 ul씩 분주하고 , 사용할 때까지 deep freezer 에 보관합니다 .


Some Cellular Organelles


•Compartments/organelles examined•Protein sequences sufficient for localization•Vital stains

Secretory Pathway:Endoplasmic ReticulumGolgi Complex

Endocytotic Pathway:Endosomes

Mitochondria

Nuclei


Transport through nuclear poresignal = basic amino acid stretches example: P-P-K-K-K-R-K-V

Nucleus


Import of proteins into nucleus through nuclear pore


Nuclear Stain:

Hoechst 33258 binds DNA


Transmembrane transport signalExample: H2N-M-L-S-L-R-Q-S-I-R-F-F-K-P-A-A-T-R-T-L-C-S-S-R-Y-L-L

Mitochondria


Protein being transported across mitochondrial membranes


Mitochondrial dye = MitoTracker Red

Non-fluorescent until oxidized

Accumulates in mitochondria and oxidized

Diffuses through membranes

Mitotracker

DNA


nuclear envelope

endoplasmic reticulum

lysosome

earlyendosome

lateendosome

Golgi apparatus

cisGolgi

network

transGolgi

network

Golgistack

CYTOSOL

plasmamembrane

Cellular components of the secretory and endocytic pathways


Entry into E.R.:Transmembrane transport signal= hydrophobic amino acid stretches

Example: H2N-M-M-S-F-V-S-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q

Retention in E.R. lumen:

Signal = K-D-E-L-COOH

Endoplasmic Reticulum

at amino terminus

at carboxy terminus


ER-Tracker Blue-White

Live bovine pulmonary artery endothelial cells

Endoplasmic Reticulum marker


Mitotracker Red and ER-blue/white


Golginucleus

From the ER, secreted and membrane proteins move to the Golgi, a series of membrane-bound compartments found near the nucleus


BODIPY-TR ceramide

Golgi marker

Ceramide = lipidWhen metabolized, concentrates in the Golgi

Red fluorophore


Golgi (ceramide)

DNA (Hoechst)

Cultured Epithelial Cells


Golgi (ceramide)

Lysosomes (LysoTracker)

DNA (Hoechst)

MDCK CellsMadin-Darby Canine KidneyPolarized Epithelial Cells

Molecular Probes, Inc.


Rhodamine transferrin

Does the fluorescent green protein co-localize?


Immunofluorescence / confocal microscopy 원리


Immunofluorescence / confocal microscopy

B or T cells in suspension, adherent cells on chambered coverglass or chamberslides, cryostat sections of unfixed, OCT embedded tissue:1. wash cells 1x cold RPMI (no wash for cryostat sections).2. Fix 20 min 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4, 0.03M sucrose on ice. ** 3. wash 2x PBS/1%BSA. From now on everything can be at room temp. or on ice.4. Permeabilize 5 min RT in 0.2% saponin, PBS, 0.03M sucrose, 1% BSA.5. wash 1x PBS/BSA.6. Block 15 min 5% normal goat serum (NGS) in PBS/BSA.7. wash 1x PBS/BSA.8. 1° diluted in PBS/BSA 60 min RT; 100 l per tube or section.9. wash 3x PBS/BSA.10. Block 15 min 5% NGS in PBS/BSA.11. wash 1x PBS/BSA.12. 2° diluted in PBS/BSA 30 min RT; 100 l per tube or section.13. wash 3x PBS/BSA; (wash 1x in PBS/BSA then 2 x 10 min in Molecular Probes SlowFade Light buffer if using Slow Fade Light S-7461 to coversip); pellet cells and put up in two drops of Molecular Probes Slowfade Light antifade medium. Pipet about 15 m l on slide and coverslip. For chambered coverglass just put two-three drops in each chamber after wash.

Immunofluorescence / confocal microscopy 실험방법

Laboratory of Molecular Genetics, KNU Gene expression & Analysis.

Documents

Transcript of Laboratory of Molecular Genetics, KNU Gene expression & Analysis.