3 replies to this topic

[Jeannine]

Hi all,

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!

Cheers, Jeannine

[bob1]

Leaky system I would guess.  I think your shRNA is being expressed somehow, perhaps by a viral promoter in the MDA-MB-231, and this is having a massive affect on your cells, while they are already stressed by the puromycin.

[Samantha]

Jeannie,  I got the same problem...did you solve the proble or figure it out what's happening?

Bob, I dont quite get what you mean by "leaky system". could you explain a bit more on that?

Thanks.

Samantha

[bob1]

A leaky system is one that doesn't turn off properly.  Tet repressors are common examples of leaky repression.