Puromycin selection when using pSpCas9(BB)-2A-Puro (PX459) : concentration and time?

[Claire Zhang]

Hi,

I have been using plasmid 48139  pSpCas9(BB)-2A-Puro (PX459) to transfect hiPS cells (25 ug/10 million cells). I started puromycin selection 24h post-electroporation at 0.5 ug/ml, I tried both 48h and 72h but did not have any colonies survived. When I tried 40h instead, I got 4 colonies out of 10 M cells. I knew that the transfection rate of iPS is generally not very high (5% when I try GFP), but 4 out of 10 million might be a little too low.

I am  wondering if I should try lower concentration (e.g. 0.25 ug/ml) and shorter time (36h?), or try a different company?  

Thanks for any ideas/comments if anyone has experiences about puromycin selection!

Claire        

[saul kivimae]

Hi Claire,

Lower puro concentration is your best bet.

This has come up on this forum a number of times that constructs with stuff added downstream Cas9 into the same transcript either by 2A or IRES have lower activity of these downstream selection functions. It is due to the promoter not making enough of the very long transcripts.

I ended up swapping the otherwise strong promoter (CMV, Cbh) with tetO+mCMV sites and cotransfect rtTA3 to overcome this issue. It boosts back up the RNA levels and selections work like they usually do.

[Xiaobing Qing]

Hi, have you already solved your problem? I also tried at least ten times using 0.4ug/mL Puromycin, but I still could not get any puro-resistant hiPS colonies. 

Best wishes,

Xiaobing

[Neville Sanjana]

Xiaobing.... Please give the lentiCRISPRv2 plasmid a try. It has a different (more standard) version of the Puro resistance gene. You can either make virus with it or transiently transfect (as you are doing with pX459).

- Neville

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