I’m trying to transfect with CB2 receptors in mammalian cells. I bought a commercial vector with CB2 sequence, without tags, and then I cloned the ORF in the pcDNA3(+), and the cloning is supposed to be alright because I’ve sequenced the vector.
I’ve tried to transiently transfect it in 293T cell line with lipofectamine, and as positive control I transfected them also with pEGFP-N1 (separated wells). I obtained green cells, but when I analyzed by mRNA and protein the CB2 levels, I found that there was no CB2 over-expression. The transient transfection with the commercial plasmid, which is supposed ready for transient transfection in mammalian cells, didn’t work.
Could the problem be the commercial sequence (but I did a ballast and matched with CB2)? Or is it a problem with the over-expression of CB2 in mammalian cells?
Hi Esther,
Just a question, does your vector have any kind of resistance? Except for ampicillin of course. I also was wondering how much time you wait before you harvest your cells.
Hi!
Yes, the pcDNA3 vector has Geneticin (G418) resistance, for stable selection in mammalian cells, now I'm trying to select them with this antibiotic. I let the cells express the vector for 48 hours after transfection.
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