I would like to conduct a "rescue" experiment in a knock-out cell line that involves designing/cloning a plasmid that expresses my gene of interest and also confers Blasticidin (or G418) resistance to the transfected mammalian cells. The goal is to enrich my cell culture pool to cells that express my gene of interest by growing them under antibiotic selection.
I have an ampR vector that expresses my gene of interest under the CMV promotor. Is there an easy way to clone in the bsd gene? Does it need its own promotor?
Alternatively, I could use a bsd expression vector to clone my gene into, but most of the bsd vectors I have found on addgene are in viral vectors. Can someone point me to a non-viral expression vector that I could use to clone in my gene of interest?
Any suggestions from experience would be most helpful and thank you in advance for your help!
cheers,
Valerie
NOTE: I just realized that the Neo gene used to confer G418 resistance also gives KanR resistance. Is this correct? if this is the case, I cannot use G418 since (details omitted) I will be co-transfecting a KanR plasmid that I will later be prepping and selecting for in bacteria...
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