I will be using CRIPSR genome editing to modify a gene and generate stable cell line. I intend to use a plasmid with puromycin selection marker to initially screen the cells. Does anyone has experience with this? Can you provide some tips and suggestions?
Also, what would be the best approach to isolate the cells into single cell?
Do these cells grow fine from a single cell? From my experience with SH-Sy5Y cells, they grow slow and can be tricky sometimes.
Hi Shruti,
I don't have any experience with CRISPR or the SH-Sy5Y line, but you could try to isolate the cells using a cloning cylinder. Once you start selection and they form small colonies, you can pick individual colonies (~10 cells) and plate them in a larger dish. you could just repeat to get to a single cell stage.
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Have you tried Limiting dilution method which is commonly used for hybridoma development which is simple, easy, and less expensive.
I have some experience with neuroplastoma cell lines although not this particular one SH-Sy5. These cell lines are a bit sensitive to work with. When I was trying to generate stable clones out of the transfection, the efficiency of recovered colonies from single cells were extremely low. I had to seed 20 cells and 50 cells per well in 96 well plate to eventually successfully have the colony of interest generated.
You can have a try. It is not hard to do a few more plates, and you will have the in two to three weeks .
Generating single cell clones from SH-SY5 is possible but takes more than 2 weeks. You have to be patient with the cells and allow them to come through after puromycin selection before you do a single cell clonal analysis and once they do, they form single clones very well.
Hope this is helpful.
Thank you all, for your suggestions.
Kwaku, have you used these cells to generate stable cell line?
I had a quick question about puromycin selection. At what concentration do you use Puromycin for these cells? They said the range is 1-3 ug/ml. What did you use?
And, at what time post-transfection did you add Puromycin? And for how long did you leave the cells in puromycin? Thanks
Generating a Knockout stable cell line took long but it was possible.
With the puromycin concentration, 1ug/ul was okay to get a stable line but the number of correct knockout cell lines reduced so I used 2ug/ul after 48hrs posttransfection and that worked very well. My plasmid too was transient so I guess it will work at your end too.
Hope it works for you
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