I am currently trying to add a tagRFP protein to the C-terminal of my target in the Huh7 cell line. The outcome was acceptable. I got more than 40% of cells generating red fluorescent 36 hrs after transfection, and cell growth was not actually affected(cell death was not observed and growth rate seemed still). However, after several passages (to passage 4), I could barely see any positive cells.
The commercial CRISPR kit from Thermo Fisher (TrueCut™ Cas9 Protein v2 and Lipofectamine™ CRISPRMAX™ Cas9 transfection reagent) was used, and I designed the donor DNA sequence, which includes tagRFP-P2A-LoxP-Puromycin resistance gene-LoxP.
My target is theoretically related to the stemness of cells, but I don't think the potential loss-of-function of it may affect cell growth or even cause cell death because we have tested cell growth status with siRNA.
So, do anyone know why I lose knock-in cells after passage, or offer me some tips to solve this problem? Thank you so much!
First do a PCR of your cells to check that it is a loss of the tag and not just silencing of gene expression (eg. due to methylation). Check the sequence of the knock in product does not have CRISPR recognition sites for your gRNA because that could lead to deletion by persisting Cas9.
Sort fluorescent cells
Non fluorescent cells might outnumber fluorescent ones
- Badges
- Science topic