I have just cloned a gene of interest in a pBabe-puro plasmid. To test if the construct works I have transfected 293 cells but I could not detect expression of the cloned gene. Puromycin resistance, driven by its own SV40 promoter, works. Does pBabe express the cloned gene only when packaged and transduced or is there something wrong with the cloning?
Thanks in advance
i have used pBabeErbB2 to generate hela clonal cells. i used VSG and GaG/pol together with my pBaBeErbB2 plasmid and it worked well. could you elaborate more on ur challenges.
Hi,you can use your pBabe vector like a normal plasmid vector, like a pcDNA plasmid, obviously if you use it to transfect 293 cells or other cell lines, you will obtain a transient transfection.
pBabe vectors don't do well with transient transfections at all. Whenever I've tried to do that in the past, it's extremely difficult to find a single cell that is transiently transfected. I would recommend testing constructs in pcDNA3.1 first for transient transfection, then moving it into pBabe and just go forward with stable cell line generation.
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