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shRNA Lentiviral Transduction- loss of knockdown after 1-3 passages - (Apr/30/2013 )

I'm trying to produce a stable knockdown in a cancer cell line using shRNA and I've run into an interesting problem.

After carrying out antibiotic selection, I harvested mRNA from my cells and saw (initially) a knockdown of the target by 50-70% (depending on primer) through RT-PCR. While maintaining selection, I passaged the cells a few days later and the knockdown had dropped to ~33%. I passaged a second time and the knockdown was -2-20%.

In brief, my protocol is to transfect GP293 lentiviral packaging cells with pSuper plasmid+shRNA, pSuper plasmid (empty), and nothing. I harvest lentivirus 24 and 48 hours later and apply it to my cells at 10-20% confluency. 24 hours after the second infection, I replace the media with media + Puromycin (concentration determined by kill curve). 72 hours after that, the empty-virus transduced cells are all dead and I perform the first RT-PCR.

I'm confident that the transfection and transduction worked because at 3+ days after infection, the pSuper transduced cells are thriving in Puromycin and the empty-virus transduced cells are 100% dead.

I'm also confident that the shRNA sequence works since I see a specific knockdown initially. Transfecting cells with the plasmid directly also induces a ~70% knockdown.

My best guess is that a subpopulation of the transduced cells are suppressing the shRNA while maintaining resistance and outcompeting the others, which is why I see a decline over time. My PI is skeptical that this could occur so quickly however.

I haven't been able to find similar cases described online- does anyone have any advice on what could be causing this or how to fix it?



Once cells are infected by lentivirus, it should get integrated into the genome and stay there. My guess is that the infection is not 100% efficient, the shRNA or the knockdown of its target gene has negative impact on cell proliferation and growth, gradually, uninfected cells take over the population. Have you tried multiple infections to increase the efficiency?