3 replies to this topic

[investigatologist]

Hi All,

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.

Thanks,

[bob1]

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)?

[investigatologist]

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?

[bob1]

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.