HSPC transduction and transplantation

Standard mouse stem cell virus–based gamma-retroviral vectors were used to express (a) human RUNX1-ERt2 (Behrens et al., 2016), (b) human FLT3-ITD (Schmidt-Arras et al., 2005), and (c) human HHEX or mouse Emx2. Donor mice were injected with 150 mg/kg 5-fluoruracil (5-FU; Medac) 3 d before HSPC isolation. Transductions were performed as described previously (Schwieger et al., 2009). In brief, 3–5 × 10⁵ HSPCs were co-transplanted with 5 × 10⁴ spleen cells into lethally irradiated (9 Gy) B6-Ly.1 mice. RUNX1-ERt2 was induced in vivo at day 14 (pulse) or day 18 by administering tamoxifen citrate via standard food pellets (400 mg/kg; LASvendi LASCRDiets). For retransplantation assays, 5 × 10⁶–10⁷ tumor cells were injected in conditioned B6-Ly5.1 mice. In xenograft assays, 10⁵ MV4;11 cells were intravenously transplanted into NSG mice.