Cell lines selected with G418 but no overexpression of GOI - (Jul/09/2013 )
I transfected cells with empty vector and one containing my GOI. I selected using G418. However when I do the IB for my GOI there is no expression. The GOI is under a CMV promoter, while the Neomycin is under a SV40 promoter. I was wondering if anyone has had a similar experience. Could it be that overexpression of the GOI is toxic to cells and therefore they are no longer expressing from the CMV promoter? I know the CMV promoter is active in these cells as transient transfections have resulted in ample overexpression of the protein. Any insights would be greatly appreciated.
Despite selection it is still possible that the plasmid has been lost from the cells, have you made sure that it is still there (PCR, Southern blot)?
No I have not done that experiment yet. Is it possible to have the SV40 promoter being active, but the CMV promoter not, allowing selection without expression of the GOI?
Potentially, it has been found that sometimes plasmids will be selectively methylated so that the resistance gene is still active but the GOI is not.