I am growing G4 mouse ESC on MEF (irradiated) layer and I wish to infect them with shRNA (lentiviral). Post infection I want to select the infected cells with puromycin. Subsequently, assess the ESC rate of proliferation and assess the KD with western blot.
I would greatly appreciate if you could share a detailed protocol for the same.
Thank you
Poorva
Hello,
I have a very good protocol but I used it for mESCs in feeder-free conditions. I am not sure how you will have to modify it for your system. As for the puromycin selection, I transduced some cells with a "control" virus (transfected HEK293T cells with all the viral plasmids but instead of a shRNA plasmid I used a control GFP). When these control cells were completely dead (around 5 days of selection), I continued to check for the kd. If you want to keep the cells as stable cell lines, remember to re-select (for a good week) after thawing before using them as stem cells tend to silence shRNA-integrated loci.
Best,
Thaleia
- Badges
- Science method