Stable transfection of L929 cells

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ygng2002's picture
Stable transfection of L929 cells

Hi everyone,

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?