Retroviral Transduction of Murine Bone Marrow Cells

Bone marrow (BM) cells from 5-fluoruracil (150 mg/kg) treated C57BL/6 mice were extracted as previously described [17 (link)]. RBCs were removed by ACK lysis buffer, and nucleated BM cells were transduced with viral supernatants containing MSCV-RUNX1^WT/G60C-IRES-GFP, MSCV-RUNX1/RUNX1T1-IRES-GFP, or MSCV-MEK2^WT/P128L-IRES-GFP for 2 days in RPMI/20% FCS supplemented with mouse stem cell factor (mSCF; 25 ng/mL; R&D Systems), mouse IL-3 (mIL3; 10 ng/mL; R&D Systems), and mIL-6 (10 ng/mL; R&D Systems). GFP-sorted cells were used for the following serial replating assays as described [17 (link)]. Briefly, single-cell suspension was prepared and 15,000 cells/1.5 mL were plated in triplicate in cytokine supplemented methylcellulose medium (MethoCult™ GF M3434; StemCell Technologies), and colonies were enumerated weekly.