No replies to this topic

[Rae]

Hi
I work with hard-to-transfect primary human cells. I need to use adenoviral-mediated transduction to over-express genes of interest in these cells but can get acceptable gene knockdown using standard siRNA (without viral delivery). I want to knockdown expression of one gene in cells I've over-expressed another gene in. My problem is I get massive cell death even with my scrambled siRNA when I do this. I've tried transfecting with the siRNA, waiting 48h then infecting with the adenovirus and I've tried simultaneously introducing the siRNA and virus. (One of the genes I'm over-expressing with the virus causes apoptosis & in theory silencing the other gene should protect against this so it won't really work to do the viral infection first then knockdown the 2nd gene which is why I haven't tried that combination). I can use the same transfection reagent (Xtremegene HP) for both siRNA and virus & my mock transfected cells don't die. Has anyone else encountered this problem? Any suggestions or any ideas why the virus/siRNA combination is toxic?