1 reply to this topic

[michalcn]

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

[laurequillo]

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

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

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