I have made pcDNA stable MDCK cells. Selected in 900ug/ml G418 (kill plate died after 12 days), got colonies, picked them with filters, grown them up, 48well, to duplicate 12 well, western blotted and have good expression of my gene. Then grew them up to 5x 15cm plates, froze 4 down and kept one to passage 4. checked expression and it had gone. This has happened for about 6 different clones now and I am cluesless as to why.
I am still using 900ug/ml G418.
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1 reply to this topic
Posted 19 November 2004 - 07:51 AM
It's a very common phenomena that stable cell line looses expression especially if the gene is lethal. One trick is to express your protein of interest fused to a fluorescent protein such as GFP, so you regularly can FACS your cells and sustain high expression.