stable transfection problems
Posted 06 July 2009 - 06:22 PM
I try to generate stable cell lines where my protein of interest is knockdown using shRNAmiR and retroviral transduction. I've already done that in one colon cancer cell line and it worked really well, i.e. the knockdown is good. Now I just wanted to try it in a breast cancer cell line (MDA-MB-231) with the same construct and same method. But my cells don't survive the puromycin treatment, they all die after 5 days. Before I've tried the transfection I determined the best puromycin concentration (kill curve). I also transfected a controlshRNA into the cells at the same time as I've done the other transfection, and that worked fine, i.e. enough cells survive the puromycin treatment. Therefore the problem can't be the puromycin treatment but also not the transfection efficiency or retroviral transduction.
Have you any suggestions what the problem can be? The knockdown shouldn't be lethal for this cell line because several groups done this knockdown transiently using siRNA.
I'm really not experienced in this topic, so it would be great if someone can help me!
Posted 07 July 2009 - 04:29 PM
Posted 25 August 2010 - 07:00 PM
Bob, I dont quite get what you mean by "leaky system". could you explain a bit more on that?
Posted 26 August 2010 - 03:37 PM