Hi all. I performed CRISPR knockout by lentiviral transduction. Cells were grown in puromycin for a period of time, protein was extracted and cells were also frozen down. A western blot showed almost complete knockdown of my target protein. After a couple of months I cultured a vial of these cells and I am now detecting abundant expression of the target protein via western blot. I have no clue how that has happened. I have also tried culturing cells in puromycin again, thinking that perhaps the wild type cells took over the population after thawing, but even in puromycin these cells are growing well. Has anyone had a similar experience or know why this is happening?
Hi Zahra,
It might be that the mutant cells with low degree of protein disruption are taking over your culture. You might have cells with small mutations, in frame, and that is why you see the protein on the western blot.
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