Hi, I plan to knock down a gene in immortalized keratinocyte (Ker-CT from ATCC). In the spec sheet, it was mentioned that these cells were immortalized with h-Tert and CDK4 carrying puromycin as their selection marker. Theoretically, it would be safer to select different selection antibiotics, however puromycin is the only one available for my gene of interest.
Has anyone transfected/ transduced Ker-CT with puromycin as selection marker? Is the selection accurate? I had seen publications where puromycin is used as the selection marker in Ker-CT, but would like to gather more information/ feedback. Thanks!
If your cells are already resistant to puromycine, you will have no selection of your recombinant cells. You could do a cotransfection with another plasmid carrying a selection. This selective plasmid must be at a lower concentration than your plasmid of interest. You could try 1:5 or 1:10, reported to the size of your plasmid
what is your control cells (un transfected) look like when you treat them with puromycin?
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