I want to transfect an already established transfected cell line with a construct of mine. The issue is that the cell line has been previously modified with a construct carrying a neomycin resistance gene. I want to know if I can still do neomycin selection post transfection with my construct and just select for an even higher level of neomycin resistance?
Yes and No
Not for transiet transfection expression exprements
But for stable transformants yes.
Neomycin will not be able to slecet for both, genes of intrest in each newly transformes cell, with the slective power for both genes of intrest.
There will be a pool with some with one gene, some with the othere gene, and some with both genes of intrest. You could have to screen twice to four times as many but their should be some that will have both.
Unless the newly inserted gene will lower the transformants viliability too much then there may be none with both. Think of the load for the cell to carry too much.
Also when doing stable transformants i would usuly see expressing single colonies would have diffrent levels of expression, i would group into low medium and high expressors of my genes of intrest.
So you could expect your double expressing cells to have both high medium and low for both genes of intrest.
Best Wishes
I agree with Charles. However, (unless you have a reporter for expression of the second gene or do some type of cell sorting) you would probably be better off and prevent future headaches if you switched the vector to a different selection marker such as puromycin or hygromycin if possible.
Thank you Erik and Charles
I agree and the question stems from poor planning on my part. I had already cloned my construct into a neomycin selection plasmid before realizing that the transgenic line I was transfecting had been established by colony selection using neomycin resistance. I will try colony selection for a higher level of neomycin resistance and if that doesn't work will clone into a different selection plasmid.
If you haven't already planned it, I'd suggest doing a kill curve on your neo-resistant cells before trying to transfect and create a new stable pool. Your current cells might just amplify their neo resistance to survive the higher concentrations.
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