I have been using HEK293 cells for years with >80% transfection efficiency with routine transfection reagents (lipid or calcium phosphate). Recently, I started using a HEK293 cell that was stably transfected with pLenti6.2-DEST based lentivirus. The inserted gene should not interfere with future transfection of the cells. However, these stable cell lines show maximum 20-30% transfection efficiency when I use pcDNA3-based plasmids. Parallel transfection of plain HEK293 cells leads to 80% transfection with the same pcDNA3 plasmid. Does anybody have such experience? Thanks in advance.
It is possibly because the CMV promoter in the lentivirus takes up most of the CMV promoter binding proteins than the CMV promoter in pcDNA3 plasmids.
when you talk about lentivirus you mean infection, isn't?
If not I think you need to INFECT your cells rather than transfect them. This is the only way to have a stable cell line.
I think in pLenti6.2-DEST transfected HEK293 cells transcriptional machinery proteins have high tendency for the pCMV of lentiviral gene cassette, if you could use a pcDNA3 without pCMV, your transfection efficiency will be improved.
Try using transfection with a plasmid that has a different antibiotic selection marker than the originally integrated transducing vector used to generate the stable 293 cell line; you can then select out the clone that gets appropriately transfected; also the point already mentioned that better to have a different promoter for your transgene than the one used already for the first transfection to generate the cell line.
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