DNA Transfection to Mammalian Cells

Three essential tools form the basis for studying the function of mammalian genes:

1.Isolate a gene by DNA cloning

2.manipulate the sequence of a gene in the

test tube

3. The technique should be able to return

the altered gene to cells to determine the

function

Extract DNA

or RNA and prepare cDNA

with restriction endonuclease

Incorporate into plasmid with selectable marker

Clone in bacteria in selective condition

Trasfection into recipient cells with lipofection, calcium phosphate or electroporation

Grow up cells transfected cells in selective medium, and assay for expression

The first methods used for DNA transfection

1. DEAE( Diethylamine ethyl)

positively charged

enter cells y endocytosis

2. Calcium Phosphate

divalent cations promote DNA entry in

bacterial cells

Exogenous DNA is Transiently or Stably Expressed

1. Transient Transfection

DNA expressed immediately after transfection

Assay by

reporter

i.e. C.A.T. :chloramphenical acetyl transferase

RNA transcription

i.e. northern blotting

2. Stable Ttransfection

Clone selected by G418 ( geneticin) or hygromycin

may be used to obtain high protein expression by

gene amplification

Dominant selectable markers Used in transfection experiments

1.Aminoglycoside phosphotransferase(APH)

G418( inhibit protein synthesis.)

APH inactivate G418

2.Dihydrofolate reductase (DHFR):Mtx-resistant

Methorexate( inhibit DHFR)

variant DHFR resist to Mtx

3.Hygromycin-B-Phoshotransferase (HPH)

Hygromycin-B( inhibit protein synthesis)

HPH inactivate hygromycin B

4.Thymidine kinase(TK)

Aminopeterine( inhibits de novo purine and

thymidylate)

TK synthesize thymidylate

5. Xanthine-guanine phosphoribosyltransferase(XGPRT)

mycophenolic acid( inhibits de novo GMP

synthesis)

XGPRT synthesize GMP from xanthine

6. Adenosine deaminase(ADA)

9--xylofuranosyl adenine(Xyl-A; damages

DNA)

ADA inactivate Xyl-A

Specific methods used For Transfection

1. Electroporation

a brief change of electric pulse discharges

across the electrode, transiently open holes

in cells

2. Liposomediated gene transfer

liposome fuse directly with cell membrane

and delivers DNA into cells

Virus Vectors

SV-40

substitute virus gene with foreign genes

( supply virus missing gene by

cotransfection with helper virus)

infect monkey cells only

carry smaller size of foreign genes

Vaccinia virus

carry smaller size of foreign genes

DNA recombination occurs in the cells

virus replicate within the cytoplasm of the

host cells

higher level of protein expression

Baculovirus

foreign gene maybe coexpressed with

structural gene ( structural protein

expresses when infection occurs)

Developing baculovirus-insect cell expression system for humanized recombinant glycoprotein

4. Retrovirus

RNA virus ( virus genome may be integrated

into the host genome)

infect various kinds of mammalian cell lines

infection of mammalian cells by retrovirus does

not cause host death

carry -galactosidase gene

viral gene expression is driven by stronger

promoter

Genome 7-10kb

contain gag, pro, pol, env :

encode structural capsid proteins, viral protease,

integrase, and viral reverse transcriptase, enveloped

glycoproteins

gag casset antibioticsR 3‘LTR5‘LTR

gagORF polORF

envORF

Advantages of retrovirus vector

Stably traduce dividing cells

Long term transgene expression

Disadvantage of retrovirus vector

Random insertion into host cell and causes oncogenic

activation or tumour-suppressor gene inactivation

Limited insert capacity( 8kb)

Low titer

Inactivatoion by human complement

Inability to transduce nondividing cells

Retrovirus life cycle

Adeno virus

Non-envelope d.s DNA virus

Genome :36kB

Early LateITR E1A ITR

CAR receptor

pH dependent release of virus particle

Immunogenic response

gutless

Recombinant Adenovirus propagated in the cell line expressing E1 region

Adeno Associated vector

Parvoviridae family

Non human disease associate

Integrate stably into chromosome 19

Transduce mitotic and post mitotic cells

rep cap ITR 145 bpITR 145 bp

Transcription unit

Helper adenovirus

ITR ITR rep cap

293 cell

Mixed helper /r AAv

r AAV production

Heat 56oC

CsCl2 gradient centrifugation

Recombinant AAV