Hi everyone,
we have a problem with our Cre-expressing cell lines, which we use to analyze cell fusion between the Cre-expressing cells and breast cancer cells expressing a double reporter vector. Breast cancer cells possess a red fluorescence. After fusion the floxed red fluorescence is excised and expression of EGFP starts, which could be easily quantified by FACS. This system worked properly, but in the past weeks we had some problems. It seems that our Cre-expressing cells have lost Cre expression since PCR was negative. This is interesting since cells were stably transfected with a Cre-IRES-Puro vector and were selected via puromycin. Cells are growing well in puromycin (5µg/ml) media, but lack Cre expression (PCR was negative).We will now perform PCR analysis for checking for puromycin resistance mRNA. Does anyone has any further suggestions or experiences about stable Cre expression in cell lines?
Hi Thomas,
I also have had in the past some issues with puromycin resistance. For some reason, some cell lines remain puromycin resistant but have lost their expression cassette. Its not so much the Cre, but the puromycin. We do have a Ba/F3 cell line with the Cre-Ires_puromycin and this for the time being is still expressing the Cre. In other cases, we moves to an IRES-GFP construct instead and this has been a better indicator of transgene expression.
My suggestion is to either change vector with a fluorescent reporter. If possible, you could go to an early passage and try a super high concentration of Puromycin - 10 ug/mL perhaps to try and find a clone which really does still have the Cre and is not just resistant.
Good luck.
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