Hello! I have a question regarding transfection stability in MCF7 and IMR32. For my experiment I need to transfect them with a plasmid with my gene of interest and Kan/Neo-gene which confers resistance against G418, as a selection tool. And after 5-6 passages in G418-containing medium I need to co-cultivate them with some other intact cell lines (i.e. further usage of G418 on this stage is impossible).
So, my question is - how long cells can retain a plasmid after removal of selecting agent?
Hello Dmitry Lifanov ,
I think it depends on more than one factor, including the characteristics of your cell line, your plasmid, the concentration of G418 that your cells are resistant to ...
Maybe if you can, try to cultivate the transfected cells with and without G418 (in parallel) for ~5 passages : you might see a difference if the cells lose the plasmid, if you can test for the presence/absence of your gene of interest after this culture.
If the cells don't lose the plasmid after 5 passages, maybe you can go up to 10 passages or more. It depends on how long you will need to co-cultivate them with the non-resistant cell lines.
I hope I could help a bit ! Good luck with your experiments.
They lose free plasmid immediately, and selection cannot stabilize free plasmid in mammalian cells. The only way you'll keep it for multiple passages is if it integrates into the genome. This will be very rare. Most cells should die within a week or so of the initial selection. Then, rare cells with genomic integration could grow up indefinitely. These don't necessarily need ongoing selection at all, but the expression level might decline due to epigenetic silencing over many passages.
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