Triple stable transfection - (May/11/2006 )

Hey,

I just cloned some genes in the TAP vectors (stratagene) and the point has come that i need to stabelly transfect them into HeLa cells.
But there is a problem, the HeLa cells already express another gene in a stable manner, and are selected on G418/neomycin. The problem is that the TAP vector alse needs to be selected on G418. Won't one of my genes fall out? How can i keep both genes in?
Shall i do a double transfection on a stable cell line? like for example transfecting my TAP construct together with a plasmid resistant to another antibiotic (hygromycin)?

What are your thoughts...

thnx

My feeling is that since the cells are already G418-resistant, you'll have to use another drug resistance cassette. Co-transfecting in with the Hygromycin plasmid should select out your transfected cells but I don't know whether you'll get a stable cell line expressing your GOI. I guess what I'm advising is to sub-clone into a plasmid containing hygromycin resistance.

All the best,
Ceri

thank you for your reply...
i thought the same thing, so now i'm trying to clone my gene plus the tag into a hygromycine resistant vector. I hope that goes smooth

thankx again
the kittie