Hi there,
I'd like to knockin in an ~1kb reporter downstream of an endogenous gene in primary FACS-purified HSCs. We have Cas9-Tg mice for these, so I was wondering what the best way could be going forward? lipofection? amaxa nucleofector? Would you transfect guide RNA or vector expressing guide? And what concentrations of each? Thanks for your patience and help.
Hey Shalin,
I think the main problem here is that the HDR efficiency is very low, especially for big templates. Most of your cells will only have a indel at your target side. You can increase HDR vs. NHEJ a bit by using SCR7, which worked for us:
http://www.nature.com/nbt/journal/v33/n5/pdf/nbt.3190.pdf
Regarding what might be the best way to get the gRNA and your repair template in the cell I would try using a synthetic, in vitro transcribed, gRNA and electroporate/nucleofect them into your cells. Plasmid electroporation is very inefficient, same for Lipofection.
http://www.sciencedirect.com/science/article/pii/S016816561500200X
Best,
Simon
I agree with Simon, maybe a good idea is to have a reporter (puromycin) that is floxed by loxp sites or Piggybac sites so you can select your population quickly
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