hP53 sgRNA-SpCas9 Lentivirus

Thumbnail Image. MOLM-13 puromycin-resistant polyclonal cells were treated with Daunorubicin for 24 hours. The cells (5×10^5) were transduced with 6 μg/ml polybrene and 100 μl of lentivirus (either TetON-Cas9-BSD, P53-sg-Cas9-Puro (All-in-One), or Scrambled (Scr-sg-Cas9-Puro)) in a 12-well plate and spun at 2000 rpm for 2 hours. After spinning, the cells were incubated for 2-4 hours before being transferred to a T25 flask. The cells were cultured for three days, then subjected to selection with 10 μg/ml BSD or 1 μg/ml puromycin, with medium changes daily to remove dead cells. Drug-resistant cells were cultured in a small volume of medium with 15 μg/ml BSD or 2 μg/ml puromycin to obtain stable cell lines. Stable TetON-Cas9-BSD cells (3×10^5) were further transduced with 50 μl of P53-sg-RFP lentivirus (1×10^7 TU/ml) for three days, then seeded in a 48-well plate at a density of about 10 cells/well and cultured in 0.5 μg/ml doxycycline. Western blotting was used to detect p53 expression, and cells with lower p53 expression were selected for experiments. All stable polyclonal cells were treated with daunorubicin for 24 hours, lysed, and subjected to Western blot analysis. The Western blot shows p53 expression in MOLM-13 cells with different sgRNA constructs (Scrambled sgRNA, TetON P53-sgRNA, and All-in-One P53-sgRNA) treated with various concentrations of Daunorubicin (0, 0.1, 0.5, 1 μg/ml). GAPDH was used as a loading control.

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