B11 cells created by replacing the NRAS-G12D mutation in THP1 cells with a dox-inducible NRAS-G12V mutation

To study the role of NRAS mutations in cell proliferation and self-renewal in acute myeloid leukemia (AML), the human AML cell line, THP1, was modified to replace its naturally occurring heterozygous NRAS-G12D mutation with a doxycycline(dox)-inducible heterozygous NRAS-G12V mutation. The endogenous copies of the NRAS-G12D allele were deleted using CRISPR/Cas9 after a dox-inducible, CRISPR resistant, NRAS-G12V transgene was introduced into the THP1 cell line. The resulting cell line was named B11. RNA-seq data confirmed that endogenous NRAS G12D was successfully replaced by dox-inducible exogenous NRAS G12V in the B11 cell line. As expected, depletion of dox induced G1 cell cycle arrest. Interestingly, the B11 cells experienced ten-times higher expression of NRAS induced G2/S-phase cell cycle arrest. Forty-nine genes were identified as signaling responsible genes associated with high expression of NRAS. Overall design: Using lenti-vector system, a CRISPR resistant dox-inducible NRAS was introduced to human THP1 cells, which also contained a G12V mutation and GFP. Cells were treated with doxycycline and GFP positive were selected. The selected cells were then transduced with a vector containing Cas9, Puromycin resistant gene, and dual gRNAs specific to upstrem and downstream of the start codon in NRAS locus. Subclones were selected using puromycin. One of the subclones, B11, was grown out and separated into 9 plates. THP1 cells were also separated into 9 plates. 3 of the THP1 plates and 3 of the B11 plates were treated with dox at 1 ug/ml, 3 of the THP1 plates and 3 of the B11 plates were treated with dox at 10 ug/ml, and 3 of the THP1 plates and 3 of the B11 plates were left untreated. After 5 days, RNA was collected and submitted for RNA-sequencing.