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Cell lines selected with G418 but no overexpression of GOI


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#1 investigatologist

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Posted 09 July 2013 - 12:03 PM

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,

#2 bob1

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Posted 09 July 2013 - 12:29 PM

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)?

#3 investigatologist

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Posted 09 July 2013 - 12:32 PM

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?

#4 bob1

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Posted 09 July 2013 - 12:35 PM

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.




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