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Increased transcriptional activity upon retroviral infection - (Jun/22/2012 )

Dear all

I'd like to investigate if the overexpression of gene "Z" has an influence on the expression of gene "X" and "Y". Therefore, I use a retroviral system (pMX backbone (Moloney-based system), Phoenix as packaging cell line). I cloned either GFP alone into the plasmid or in fusion GFP-Gene"Z".
I observed, that if I infect my cells with the retroviral construct and analyze gene "X" and "Y" by qRT-PCR, that there is a huge increased mRNA expression compared to noninfected cells. This is due for both constructs, the empty vector as well as the one overexpression gene "Z". It seems that infection with the GFP construct (serving as a negative Ctr) is already altering the transcriptional activity of some genes. Thus, the effect of the overexpressed gene will be diminished... (normalization against houskeeping genes 40s or 60s) Attached Image
I guess this is due to the 5'LTR of the plasmid, which do have some transcriptional enhancers and regulatory elements. However, it should be feasible somehow... Is one of you using retroviral infections for a similar purpose? How do you cope with that problem?

Thanks a lot for your help!
Cheers, Daniela


I'd still need some suggestions / help... Thx a lot!


Check the protein!