Hi all,
I have generated a few cell lines and I keep getting this mixed problem.
I transfect the cells with 50-500ng of the plasmid (CMV promoter and SV40 for the selevtion marker).
I add selection marker and dilute the cells to generate a single cell colony.
after expanding some of the single cell colonies that are positive, I see that still only some of them express the desired gene (it is a fluorescent protein). Even after sorting with FACS, the enriched population still is not homogeneous.
Am I missing anything?
Thank you
Most likely the expression LEVEL is different. For a fluorescent protein to be visible, you need a lot of it, while for markers (like puromycin resistance or other selection tools) even a small quantity is enough. Assuming that you use a system with random integration into the genome, some of your transfected genes will end up near some "silence-prone" regions that will hinder transcription.
It's a hypothesis that I've heard from experts about my plasmid. I don't think anybody has a better explanation.
Hi! Are you using the selection antibiotics when you are expanding your single cell colonies? This could be a major reason for the heterogeneity.
Thanks Yulia, I guessed it probably some thing with gene silencing but still I wonder how can that be solved :)
Thanks Vatsal, I am using the selection marker all the way through.
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