We tried to knockout a gene of our interest by CRISPR/Cas9 using lentviral approach. We transduced cells with lentivirus containing Cas9-gRNA-puromycin, selected with puromycin for 5 days, then did the serial dilution in 96-well plate in order to have single-cell clone. After expansion, we did WB using antibody recognizing 14 first aa of the protein of interest and found some potential KO clones. Then, we did sequencing the amplicons arounnd gRNA sequences, and found these KO clones always heterozygous that both contain 7-nucleotide deletion leading to a truncated protein which contain the 14 first aa recognized by the antibody used for WB. We amplified the KO clones (5 passages) and surprisingly found our protein of interest reexpressed by WB. Do anyone have the same problem and how can it be explained?
PS: Our protein of interest seems to be important for the cells. Knockdown of that protein inhibits strongly proliferation.
Thank you very much,
If as you say full expression is important for proliferation, then your heterozygous knockouts would be under strong selective pressure to upregulate expression of their remaining copy. I think it's normal and expectable that they would figure out how to do this within a few passages.
I think the solution here would be to use tamoxifen inducible cre plasmid. This way you can proliferate the culture from the single-cells and then deactivate your protein of interest having a large number of cells to work with to study the phenomena with the absence of the protein of interest, keeping the inducible cells in the culture and stock for further work.
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