Hi,

I am trying to perform KD of my protein using lentiviral inducible shRNA, am working with U2OS, A549 and MDAMB231 cell lines. As a viral packaging control I used non-inducible lentiGFP, which was positive. Later I did puromycin selection, until all WT cells died, cells were kept frozen for two weeks in -80 deg. After thawing them I did another round of selection, most of the cells survived. For DOX control I have inducible lenti teton DsRed yet even after 72hr of DOX I had no DsRed expression nor KD. I tried 2 different DOX derivatives, tried using fresh DOX (one that hasn't gone through freeze-thaw cycles) and tried different DOX concentrations (100, 150, 200, 500, 1000ng/ml) but the result was the same.

Does anyone have a suggestions or experience with thawing selected cells and seeing affected KD?

thank you