Hi all, I have many stable cell clones and I have found that (by IF) only a subpopulation of cells express the protein of interest (POI) in each clone. This is how I generated the clones:
1. I used CRISPR-Cas9 to knock out the POI in WT cells.
2. The pool was then sorted into single cells on a 96-well plate > KO was verified by western > verified single cell clones were kept for use.
3. To one KO single cell clone, I re-introduced the gene of the POI (under a tetracycline-inducible promoter) by lentivirus > puromycin selection > single cell sorting > verify protein expression (under doxycycline induction) > verified single cell clones were kept for use.
While my intention was to end up having stable cell clones, within which all cells can express the POI near physiological levels (by adjusting doxycycline levels), the reality is that not all the cells within my stable clones express the protein, and I am desperate for possible explanations.
Some colleagues have suggested
1). Gene silencing over time. However, the cells are not particularly old (~20 passages before being discarded).
2). Doxycycline induction efficiency, as some of them have confirmed doxycycline does not work equally on all the cells (e.g., not entering some cells, faster metabolism in some cells, ...), even within a stable cell clone.
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