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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
with restriction
endonuclease
or RNA and prepare
cDNA
Grow up cells
transfected
cells in
selective
medium, and
assay for
expression
Trasfection into
recipient cells
with lipofection,
calcium
phosphate or
electroporation
Incorporate
into
plasmid
with
selectable
marker
Clone in
bacteria in
selective
condition
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
gene amplification
expression by
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

5‘LTR
gag
casset
gagORF
antibioticsR
3‘LTR
polORF
envORF
Genome 7-10kb
 contain gag, pro, pol, env :
encode structural capsid proteins, viral protease,
integrase, and viral reverse transcriptase, enveloped
glycoproteins
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
ITR
Early E1A
Late
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
ITR 145 bp
rep
cap
ITR 145 bp
r AAV production
Transcription unit
ITR
ITR
rep
cap
Helper
adenovirus
293 cell
Mixed helper /r AAv
Heat 56oC
CsCl2 gradient centrifugation
Recombinant AAV