CRISPR-Cas9 has been used to knock out a gene in breast cancer cell (MCF7) by using two different plasmids. One plasmid has a puromycin selection marker and another one has a GFP selection marker. Although we use the same gRNA to knock out the gene, we have seen different cell's behavior after the gene was knocked out. Is there any idea why the difference? Does puromycin/ GFP markers affect the cells? I appreciate any help.
If you isolated clones, it would most likely be just due to natural variability between the clones. This isn't necessarily related to your crispr modification. If you isolate clones by plating the cell at extreme dilution, you'll see some pretty different colony shapes, cell shapes, growth rate, etc, in the absence of any gene modification. It could be just because you're isolating natural diversity already present in the cell line.
Thanks John. Is there any specific guide by which I can choose the best clone after knock out?
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