I established stable cell lines by gene knockout using lentivirus (GFP expression vector, CMV promoter, Puromycin selection) under optimal antibiotic drug selection. I used viral titre generated by a lab member 3 years ago and stored in -80deg.
The gene was interest was knocked out and determined by western blot. However after 6-8 passages, the cell are loosing GFP expression but my gene of interest remains knocked-out, as determined by western blot again. Are these cells good enough to start in vitro studies or do I need to select single colonies again from my earlier stocks?