How is the GFP being expressed? is it an IRES, 2A, fusion with shRNA, something else?

I find when I do lentiviral transduction, I often get a lot of integrations that are not the complete viral RNA that you've inserted into the plasmid (I've checked this by looking at cassettes with double markers and finding that a lot of transduced cells are not double positives). This is presumably because of recombination weirdness happening in the host cell. So, from a technical standpoint, it could be that cells with truncated cassettes caused by the GFP being partially recombined out might end up with way better expression of the shRNA?

Biologically, there could be a feedback loop working. If you have very high knockdown, the production of your protein could be upregulated by the cell. What you observe as "high knockdown" might actually be an intermediate knockdown that doesn't trigger this activation response.

Those are both total guesses though.

Hi Govinda,

    Thanks for your reply. I used the pLK0.3G vector purchased from addgene. The GFP is EGFP. This vector used U6 as promoter, and the shRNA was inserted before EGFP using restriction enzymes. Theoretically, the expression of the second protein (GFP) should be weaker than the first one (shRNA). So, it should be that the more the GFP the more the shRNA. However, my results showed that this is not the case. When you did the lentiviral transduction, what vector did you use?  Did you find integrations with markers only? I agree with you that there may be a feedback loop. I will do literature search for some references. Thanks for your valuable suggestions