The effect of two lysines in PML/RARa on mouse haematopoietic stem/progenitor cells

Analysis of mHSPCs infected with PML/RARa(WT) or PML/RARa(2KR: K227R and K360R in RARa moiety). We used RNA-sequencing to detail the global programme of gene expression in PML/RARa(2KR)-overexpressing mHSPCs. Results provide insight into the reduced clonogenicity caused by PML/RARa(2KR) Bone marrow cells were first obtained from the femurs of 5-fluorouracil-treated C57BL/6 mice, and then mHSPCs were selected from bone marrow cells using the Easysep Mouse Hematopoietic Progenitor Cell Isolation Kit (Stemcell Technologies). mHSPCs were cultured in a six-well plate and exposed to concentrated lentivirus (pMSCV, PML/RARa(WT) or PML/RARa(2KR)) in the presence of 6 µg/ml of polybrene. Infection was obtained by centrifugation of the cells in viral supernatant at 1,800 rpm for 40 min. The cells were cultured for 24 h at 37 °C. The following day, the medium was removed and replaced with fresh culture medium. Transduced mHSPCs were sorted and collected for RNA extraction and RNA-sequencng.