Cbh promoter vs CMV promoter

[Hung Nguyen]

Hi all,

I noticed that the recent espCas9(1.1) plasmid uses Cbh promoter for the expression of espCas9 protein. Does anyone have experience with this particular promoter? How strong and stable is it compared to other strong promoters such as CMV and CAG? 

Thank you,

Hung

[Matthew Thompson]

The other Zhang lab plasmids also use this promotor. I've used PX459 V2.0 and PX462 V2.0 (puro resistant plasmids), and gene editing is very effective. There's a brief discussion of the CBh promotor in Box 3 of their Nature Protocol paper.

[Mark]

Matthew, do you use puromycin to select?  I think others on this forum reported the resistance was not that good and many cells died. I am curious are you using transfection or electroporation? Thanks.

[Matthew Thompson]

I transfected PX459 V2.0 and an ssODN into HT1080 and U2OS cells with Lipofectamine 3000, treating with puro at 2.5 µg/mL for 72 hours. This short selection period was used since I wasn't sure of the transience of the puro-resistant plasmid retention. The puro concentration was chosen after performing a cytotoxicity profile over this time scale.

Many transfected cells did die from selection, but after a week of outgrowth in puro-free complete medium, HT1080 cells recovered. U2OS cells grew much more slowly, althought they did eventually recover. From there I checked activity with T7EI and verified my SNP by RFLP.

[ankitj...@biotechtu.edu.np]

Hello matthew,

I am also using the same vector for transfection in Hela cell line with lipofectamine 2000. I want to ask you that what was the knockout efficiency in your experiment and did you perform western blotting to check the protein expression for complete knockout?