Lentiviral transduced cells loose insert?

Andres Fernandez Gil @Andres-Fernandez-Gil

18 October 2023 1 2K Report

I have infected iPSCs with a lentivirus and sorted GFP positive cells. But after just a few rounds I am finding cells without GFP in culture. Had anyone seen this? Maybe cells are segregating inserts into only one of the cells after division? I think that this could be the problem, but never saw this before.

Mauro Lago Docampo

What is the promoter? In our experience, If the plasmid has CMV or another viral promoter it tends to be silenced after a few passages.

Also, what passage are the cells? lentiviral cargo insertion is random in the genome, if the cells are early passage the epigenetics may also change.

Piggyback insertion and puromycin have been what worked the best for us.

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