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
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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.
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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