Currently I work on endometrial cancer and I work on the gene PAK1. I had prepared knockout in endometrial cancer cells like Ishikawa but when it come to making knockout cells in AN3CA I have a lot of problems. Ideally the doubling time of the cell line AN3CA is 54 hours.
CRISPR-Cas9 system uses a mixture of four different plasmids from Santa-Cruz and they are the commercial plasmids purchased from the company. In spite of that I have difficulties in making knockout cells.
1) I use Fugene transfection reagent at the ratio of 2UG (micrograms) plasmid mix: 6UL(microlitres) Fugene.
2) I transfect it in Opti-MEM media and keep it for 6 hours and add the complete media followed by media change after overnight incubation with the complete media.
3) Plasmid has a GFP marker and its used for single cell sorting.
The problem is post sorting, the cells form colonies but the colonies don't grow in wells of the 96 welled plate. Why is this happening ? Is it because of sorting or because of the gene. I had looked into the literature where they had prepared knockouts in the same cell line (AN3CA) for other genes but they had got knockouts but for my case I am not getting a single knockout in this cell line. Please help me out in this matter..
Hi doctor
I think this article may help you
https://pubmed.ncbi.nlm.nih.gov/31039627/
Sometimes there's a mistakes with handling .
Sorting is pretty harsh on cells - to the extent that some don't survive the procedure. Could you use antibiotic resistance to select the transfected cells?
Could you also collect non-transfected cells and see if they grow normally?
What is your transfection efficiency? If not too low, maybe I would consider trying limiting dilution method. It's way less stressful for your cells in comparison with single-cell sorting with cell-sorter...
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