MSCV mir30 retroviral vector problem - (Mar/08/2007 )

<_<Hi!
I have a problem. I have produced viruses in my 293T cells, and Phenix A cells. Then when I'm infecting my target cells I can not see any expression from the vector. The construct has incorporated GFP ( expression from SV40 promoter). Almost all of the packeging cells were very green, but almost none of target cells!!! What could be the problem. I inserted shRNA into my vectors, and I have confirmed the integrity of the vector, and I have also sequenced everything. The data at that point look very good. I was soo sure that everything should be fine, but now is 28h after infectionofmy target cells, and nothing they are blank like...... Please help me with that?

You have to wait for 3 days for optimum expression with many viruses (some even longer) unless you are using SFV. Also does your promoter work in these target cells. This could influence the delay in protien expression.

Check cells after 3 days, I am sure you will find GFP positive cells.

Thank you I will wait then, and hope I will fing some positive cells:)