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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