Hi everyone!
I need to stably and randomly integrate two plasmids into HEK293 cells. No CRISPR required. We need random integration.
My current design is that one plasmid carries G418 res gene and the other - blasticidin res gene.
So the question is has anyone had good experience in generation of double stable cell lines in one step? I understand that double stable cells could be generated via sequential selection. But it is more time/resource consuming.
Double selection clearly works (Tried with puro/G418, not with Blast/G418); they all work on protein translation, but they're different drugs catabolized by different enzymes. Note however that HEK293T are resistant to G418...so you'd need another selection marker.
Make sure you allow for a couple of days for the selection markers to express properly. Problem is that, depending on your expression system (promoter vs bicistronic), you will get (some) selection with X without expression of Y (random insertion); just validate expression of your RNA and (ideally) your protein of interest. And don't forget your death curves (combined and singly).
Good luck
Double selection clearly works (Tried with puro/G418, not with Blast/G418); they all work on protein translation, but they're different drugs catabolized by different enzymes. Note however that HEK293T are resistant to G418...so you'd need another selection marker.
Make sure you allow for a couple of days for the selection markers to express properly. Problem is that, depending on your expression system (promoter vs bicistronic), you will get (some) selection with X without expression of Y (random insertion); just validate expression of your RNA and (ideally) your protein of interest. And don't forget your death curves (combined and singly).
Good luck
Thank you, Sebastien! Your answer sounds encouraging! Sure, I will do death curves, it's on my list. In fact, both plasmids will not express anything except the selection markers, so there's no problem with that.
I want to emphasize Sebastien's point that 293T cells are G418 resistant. Be sure you have 293 cells and not 293T cells.
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