Transfection control dCas9-KRAB cells

Gene-editing Discussion
1

I am looking to knock-down an miRNA using the dCas9-KRAB-TagBFP line. I’d like to ensure that my transfection of the cells with the sgRNA is successful by including another constitutively expressed fluorescent protein. Has this been accomplished before with these cells? I am concerned that the cells may become too stressed and will die. Any recommendations?

Thank you!

2

Hello @en2b,

Thanks for the question. In our experience, transfection with another constitutively expressed FP does not stress the cells too much and any stress is due to the transfection process itself and having plasmid DNA in the cells, rather than what is getting expressed. We believe including another constitutively expressed FP should work. A CAAGS driven FP always works well for us in these situations where relatively little plasmid DNA can be transfected while still getting outstanding signal.

Please reach out if you have more questions.

Janani