Selection of stably-transfected mammalian cells - what to do and why ? (Nov/22/2005 )

Hi all,

I'm quite new in the cellular biology field and I have few questions regardings selection of stably-transfected mammalian cells.

I basically want to co-transfect 2 plasmids (pcDNA3) into a rat fibroblasts cell line. Thoses plasmids both contain the ampicillin and neomycin resistance gene. I am using the Lipofetamine2000 reagent and I want to be sure that both plasmids are actually going to be in my cells. I thus need to select my cells using antibiotics. But as the plasmids are similar, how can I do? If I use only Geneticin (G418), I will not be sure that my cells bear both plasmids, am I right?

In that case, is it possible to use ampicillin + geneticin to select my cells as the plasmid have both genes. I know that usually people use amp to select transformed bacteria but I haven't found any information about why I could not use amp to select mammalian cells. Is it because mammalian cells express an ampicillin resistance gene already?

I would really appreciate any usefull information !!

Thanks a lot !

OK, I guess my question is kind of unfounded, right? I figured out that as my plasmids are similar, selecting with both antibiotics is not going to help me since both contain the resistance gene...

So I guess my only solution is to switch one gene of interest in another plasmid containing another selection gene, different from neomycin.

Still, if somebody has an answer to the Ampicillin question, meaning, can it be use for mammalian cells or not, and why, that would be helpful.

Thanks.

As amp resistive protein (lactamase???) is only expressed under prokaryotic regulatory region it will be made only in Ecoli (Just as Selective Marker for Transformed Ecoli). Not in euk-cells andso can't be useful in Euk-system.
Even if being expressed in Euk- cells, it is not toxic towards the Euk-Cells andso no question of selection.

Ampicillin is not toxic to mammalian cells in normal doses. It is pretty comparable to penicillin that most people use together with streptomycin to keep cell culture bacteria free.

As a second marker, use puromycin or hygromycin resistance.

OK, that helps. I'll try to find a constitutive mammalian expression vector with a puromycin resistance gene.

Thanks for the advice !

M-kun