stable cell line - selection after retroviral infection (Jul/08/2010 )

Hello,
I transfected phenix cells with pBABE plasmid, collected the virus and infected my cells of interests.
I selected with puramycin until the blank were all dead and collect the cells for RNA and protien.
I expected to see overexpression of my gene of interest but it didn't happen.

I sequence the vector and saw that my gene at least by sequence is there

what are the options for this?
Is there any solution for this problem?
I assumed that my cells are resistance to pura because of the infection but are do you think that they got only the resistance without the constract?

please help!

Thanks
Michal :lol:

-michalcn-

michalcn on Jul 9 2010, 04:53 AM said:

So you couldnt see it nor in RNA neither in protein. Did you check your pBABE in a transient transfection? without any virus, just transfect the plasmid in your cells. I would try that to check if I am able to produce my protein. If not, then I would think there is something wrong with the construct

-laurequillo-