Trouble producing stable cell lines
Posted 01 April 2012 - 01:10 PM
I need to produce stable PC-3 cells overexpressing my gene of interest. I have cloned a gene of length ~2kb driven by the CMV promoter in pCDH vector and I produced the lentivirus from 293T cells using my vector, pVSVG and pSPAX2 (for packaging and signalling). I infected PC-3 cells with this virus but I couldn't detect any GFP signal in the cells. So, I repeated the infection with the same virus in HeLa cells as they are easily transfected. But I got the same results in HeLa cells as well. Virus produced for a protein knockdown in my lab using the same conditions seems to be working fine. I am transfecting my vector transiently in PC-3 cells this week, to check if my plasmid is okay.
I was wondering if this has something to do with the promoter in the vector or is it just a simple problem of optimization for this vector ?
I 'll appreciate any help right now.
Posted 02 April 2012 - 09:05 AM
If you used a cDNA clone with 5' and 3' UTR there could be regulation occuring in those regions.
I would check your plasmid integrity, by a restriction digest and PCRing the plasmid. Plasmid integrity will be easy to assess then
Posted 11 April 2012 - 04:56 PM
I did the transient transfection, and was very happy to see fluorescence in my cells ! But when I started selecting with Puromycin(1 ug), almost all the cells died. I reduced the concentration of Puromycin to 0.5ug to see if any of the cells would still grow, but there was almost 95% cell death. I don't know what really is going on as these cells are all expressing GFP after transfection, but none of them are resistant to the antibiotic. Could someone explain this please?