false positive clones URGENT - resitant to G418 but not expressing protein (Mar/28/2007 )

hey,
I have the same problem with Hygromycin too,
could you find the solution? let me know if find it.
thanks

Hello!

I've had the same problem! I’ve tried to establish stable cell lines expressing a gene of interest by transfecting and selecting HeLa cells with G418. First I’ve made killing curve to determine the best concentration that killed the cells after 1 week (500µg/mL) and then started the transfection. After two weeks of selection, my control cells where dead, so I made a western blot to control the expression of my protein of interest and there wasn’t any expression!!?? I thought maybe it was an effect of the G418 on the expression of the transgene but 2 weeks after there was no more expression… Or maybe a random cut in the plasmid that often happens in my insert (3,2 kb!).

So I’ve decided to use a retroviral transduction (insertion with LTR and not random cut!). I’ve cloned my insert followed by an IRES-Neor, made the infection and selected the cells during 2 weeks (control cells dead) and this time again, no expression of my protein even after 2 weeks without G418…

So I really don’t understand what happens in my cells. Can someone help me please?

Thanks

sorry, i'm new to cell culture. what is meant by a "clone". how would one select such clones and what do these look like after you have made a stable cell line? thanks.

Well I did, the clones were positive but were just expressing minimal amounts of the protein and therefore didn't stain well.

I don't know if this is applicable to the work, but you could link the protein together with the resistence gene via a 2A-self cleaving peptide. That way you have one promoter driving expression of a fusion gene, which once expressed cleaves itself into two individual proteins.