gene targeting in cell type other than ES

[Martin Kennedy]

In article <fodde.16.000E33D1 at ruly46.leidenuniv.nl>, fodde at ruly46.leidenuniv.nl (Riccardo Fodde) writes:
> Hi everybody,
> 
> I would like to ask for information on gene targeting in EBV-transformed 
> celllines.  Has anybody tried this and how to get single colonies?
> Also I would like to know if it is possible to target primary fibroblasts 
> without having to immortalize them first and still have enough passages left 
> to do something with correctly targeted cells.

It is not easy to get good transfection efficiencies in EBV lines, so your
targeting success may depend on finding a method that gives you enough 
resistant cell lines.  I've had little luck with electroporation, which
gives good transient transfection efficiencies but very few stably 
transformed lines.

Make sure you use hygromycin as a marker, not G418, as EBV cells are
naturally resistant to about 1mg/ml G418.  You won't get colonies, as 
they are suspension cells, and you will have to plate out into 96 well-
plates.  

These refs might be of some use, though there are probably plenty of 
others:

PNAS 88, p4708	Accili et al., 1991  (KO in mouse 3T3 fibros)
PNAS 90, p3705  Xu et al., 1993    (KO in murine pre-B cell line)		
PNAS 90, p9832  Zhen et al., 1993  (KO in myeloid leukemia line)
Genes & Dev 6, p928  Kim et al., 1992.  (KO in erythroid line)

Hope this helps
-- 
Cheers,

Martin

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