Previously, I successfully knocked-in a transgene into HEK293 cells by using paired nickase CRISPR system. Now, I am trying to do it with human primary fibroblasts (nHDF). I co-transfected 3 plasmids by using Lipofactamine3000 and transfection efficiency was too low (10-20 cells in 80% confluent well in to 6 well plate). I applied antibiotic selection and hardly 10-20 cells survived (probably those with successful transfection). After making them confluent, I failed to see the GFP signal again. Is it possible that positively transfecetd cells got antibiotic resistance without the expression of transgene? If so then what mechanism undergone (any reference)?
My transgene is not expressing (no GFP). Is it worthy to confirm knock-in by junction PCR? Any way to improve knock-in? Please advise me!
As you're using 3 plasmids for transfection, the transfection efficiency is very critical. You need to have high co-transfected events. In other words, in order to have editing event, all 3 vectors must be presented in cells. I recommend to use electroporation approach.
Other thing you should keep in mind, if your nHDF proliferation rate is low, the KI efficiency is also low!
Good luck!
Thank you Dr. Ngoc~! Do you have any views about the non-expression of GFP with positive selection of cells after antibiotic treatment? How cells are surviving under the selection pressure without transgene (GFP) expression.
Hi Nasir,
I definitely suggest you to use electroporation. It's much more efficient than transfection for knock-in. Using an ssODN (139nt) or the Easi-CRISPR approach will improve your knock-in, as well.
Cheers.
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