TetOn Knockout Kit

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HEK293FT cells were transfected with TetOn-SpCas9-puro (one nuclear location signal, NLS, at C-terminus) or TetOn-SpCas9-2NLS-puro (2NLS at N and C Termini) for 16 hours before adding 0.5 μg/ml Doxycyline for additional 24 hours. SpCas9 is expressed most in the cytosol and minor in the nucleus and one NLS is enough for its natural expression and nuclear location, because 2NLS at the N and C termini of SpCas9 did not increase its nuclear location but rather reduced its stability. This result indicates that overexpression of SpCas9 in the nucleus is toxic to the cells (seen in round up cells) and normal function likely due to genomic stress. Therefore, using TetOn Cas9 system to control Cas9 expression temporally is essential for succeeded knockout of target gene and maintenance of cell normal function. The TetOn SpCas9-puro or BSD lentivirus allows you to establish stable TetOn-SpCas9 cell lines with puromycin or blasticindin selection. These cell lines can be transiently or lentivirally transfected with sgRNA oligos/library or sgRNA-expressing lentivirus under Doxycycline control to get stable knockout pool or clones for target gene/drug screen. LipExoGen offers purified high-titer transduction efficiency lentivirus of hU6-sgRNA-GFP,RFP with or without selection for quickly getting your desired functional knockout cells. These results demonstrate that the all-in-one TetOn cDNA system in LipExoGen works very well even for expressing large size proteins such as SpCas9.

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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