stable transfection

[Andreas Elsing]

> Is it true that it is better to linearize the vector for
> stable transfection protocols and if so where should
> we cut the vector?

Yes it is. The uptake and integration efficiancy is meant to be much
better when transfecting a linearized vector.
A suitable cutting site depends on your fusion partner. Of course it is
not a good idea to use an restriction site that is present in your fused
protein or in the EGFP...
In other words find a unique site within a part of your vector that is
not needed for eucaryotic gene expression. I did two N-terminal fusion
constructs using pEGFP-N1 and the Afl II site (just 3' from the SV40
poly a) worked quite well for me. After stable transfection
(electroporation) and G418 selection in 293-cells i got nice fluorescent
clones suitable for FACS and confocal IF.

Best regards and good luck

A. Elsing
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Andreas Elsing
Max von Pettenkofer Institut für Virologie
Genzentrum
Feodor-Lynen-Strasse 25
D-81377 Muenchen

Tel.: +49 89 2180-6856
Fax.: +49 89 2180-6899

Please visit our Homepage:
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