Hi,
I have generated some lymphoma cell lines and have been able to transduce them to high levels (>60%) using lentivirus expressing GFP.
I have tried titrating these cells with a vector that expresses gRNAs for a future CRISPR screen (the cells don't express CAS9 yet). I am transducing by spinnoculation and treating with puromycin at 1ug/ml (which I have titrated against the cells). When I perform the titration, I get a nice curve. My problem is that by comparing the numbers that I get from my no virus control or even cells that have been transduced but not undergone selection it would appear that I am only able to transduce ~10% of my cells (even though I know from GFP-expression that I can get much higher).
Is it possible that the puromycin treatment is causing the cells to stop proliferating for a short time (ie one or two days)? Or can anyone suggest another reason for this?
Many thanks
Nick
Yes, i think it will effect the transcription or translation process because as we known, Puromycin is an aminonucleoside antibiotic produced by Streptomyces alboniger.
It specifically inhibits peptidyl transfer on both prokaryotic and eukaryotic ribosomes.
Hi,
in fact as for any kind of selection you will see a lag pahse. In my hands Puro selected cells took at least a week or so to recover. This is due to the selection as well as the stress coming anyhow from the transduction etc. Further due to the selection it self you reduce the number of living cells and many hermatopoietic cells have issues to proliferate in low densities. So I think this is a behavior to expect
Sven
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