I have troubles with knock downs of different target genes in different cell types. They all behave the same.
Protocol: Spinning protocol for lentiviral treatment with polybrene and puromycin treatment after 2 days.
Used cell types: Jurkat, CEM and adherent carcinoma cells
Used targets: 3 different targets (some of the shRNAs are validated by Sigma)
The problem is as follows: After the lentiviral transduction (on day 2), I harvest some cells for FACS or western blot analysis. I always see a knock down of at least 50 % of my target genes/proteins compared to wild type [wt] and control (scramble shRNA) cells. I now begin with the puromycin treatment (Depending on the cell type with up to 5 µg/mL puromycin). Untreated wt cells die, lentiviral cells stay alive, like they should behave.
The problem is now, when I test them again on day 7 after lentiviral transduction in FACS or western blot, the knock down, compared to wt and control cells, is gone again. I cannot see any differences! The cells are resistant to the puromycin treatment, since they don't die, but wt cells do.
As I said: Some of the shRNAs are validated and I have seen data in other publications, indicating that they work fine. Some of the constructs also already worked in our lab for other cell types. Maybe I am doing somethign wrong? But where can be a problem, since the cells are resistant?
Maybe you can help me?