I am working on stably transfecting L929 cells. So my gene was cloned into a pCMV6-XL6 vector. It is a vector that uses CMV promoter and SV40 for neomycin resistant gene. I used Lipofectamine 2000 for the transfection. 2 days after transfection, cells were treated with 400ug/ml G418 for about 10 days. Colonies were formed on the transfected plates but not in the untransfected plate. I picked colonies and expanded them, then checked protein expression by western blot. However, there were no protein expression on the 12 colonies that I had picked. I checked the sequences of my plasmid, it was fine. And transient transfection of 293T showed a strong protein expression.
My question is why my protein was not expressed but the neomycin gene was expressed in L929? What else I can try to make it work? Does anybody have experience with L929 cells?