Stable Cas9 expressing hESCs H1
I am in the process of developing a CRISPR screen which requires Cas9 expressing hESCs. I have been following the protocol outlined in the Joung et al. 2017 Nature protocols paper. However, I am having a lot of difficulty producing a Cas9 expressing H1 cell line. I have transduced these cells in parallel with a GFP lentivirus and selected with puromycin just fine. But with the Cas9 H1 cells after puromycin selection I can not detect expression of Cas9 via IF.
Has anyone had issues transducing H1 cells with a Cas9 lentivirus? Any suggestions are very welcome.
I have not had any issues transducing Cas9 into H1 hESCs. My guess is that maybe your Cas9 lentivirus is lower titer because the vector is larger. Did you have controls to confirm that puromycin selection was successful for the Cas9 transduction?