Does someone have experience in generating crispr-KO luminal breast cancer cell line e.g. MCF7/T47D?

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Didier Fesquet

Instead of cell sorting ..use pX...puromycîne , transfect your cells, the next day add puro. When the non transfecter cells are dead. TRYPSIN the puro résistant cells, and plate few 10cm dishes, With 1000, 2000...ie à range if cell number allowing colony grow.

from the remaining puro résistant pool, perform your favorite assay (WB, PCR, T7assay, surveyor ) ..to évaluate the KO efficiency...

it will help you decide how many clones you will analyse..

when your colony have reach a reasonable size..just take them out from the plate with colony ring, cloning disk ..and seed 24wells plate...subculture and perform your favorite assay to check for KO...it is really straightforward .i have tried cell sorting but give me too many clones to screen..and lots of mortality...but did not tried to optimize.

Wen An Pan

Hi Didier,

Thank you so much for your suggestion. I will try your protocol next time. Thank you!

Best,

Wen An