I have been trying to stably knockdown a gene in mammalian cells using viral supernatant from HEK293FT cells. Although I have found transfection efficiency to be good enough multiple times, after a few weeks of virus transduction in the cells and confirming the knockdown, the gene is seen to be re-expressed in spite of it being a lentiviral construct.
Any suggestions on why this should happen and what can be done?
Dear Madhura Ketkar
This is a very complex problem and requires extensive examination and may ultimately depend on the function of your gene. However, may I ask, did you perform an antibiotic selection of transduced cells? Even if, it may lead to a polyclonal population as different viruses will integrate into different locations leading to different transcription rates of the shRNA. However, a monoclonal selection can be performed to get a uniform population of cells for your experiments. Although asking for your hypothesis might not be a good idea, but it could also be related to the function of the gene as with time the cells expressing your gene having proliferative advantage (normal) could have been enriched. Did you notice any behavioral changes in cells since transduction as compared to normal cells?
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