Blood. 2006 March 1; 107(5): 2170–2179.
Retroviral overexpression of Ezh2 in HSCsPrimary bone marrow (BM) cells were transduced as previously described with some adjustments.35 Briefly, BM cells were extracted from mice (CD45.1) given intraperitoneal injections of 150 mg/kg 5-fluorouracil (5-FU; Pharmachemie Haarlem, Haarlem, The Netherlands) 4 days earlier. Cells were cultured for 48 hours in StemSpan (StemCell Technologies, Vancouver, BC, Canada) supplemented with 10% FCS, 300 ng/mL PEG-rrSCF (Amgen, Thousand Oaks, CA), 20 ng/mL rmIL-11 (R&D Systems, Minneapolis, MN), 10 ng/mL Flt3 ligand (Amgen), penicillin, and streptomycin. Viral supernatant was harvested 24 and 48 hours after transfection of ecotropic Phoenix cells and inoculated in 6-well plates that were coated with 50 μg retronectin (Takara, Kyoto, Japan). Plates containing the viral supernatant were spun for 1 hour at 800 g at room temperature. Four hours later viral supernatant was removed, and 7.5 × 105 cultured BM cells were inoculated per well together with 4 μg Polybrene (Sigma). At the second transduction 2 μg Polybrene was added. Four days later transduction efficiency was determined by flow cytometry (FACSCalibur; Becton Dickinson, Palo Alto, CA).
