All animal studies were approved by the Animal Use and Care Committees of the Center for Blood Research and Harvard Medical School. Balb/c mice (The Jackson Laboratory or Taconic Farms) from 6 to 12 wk of age were used in all experiments. In some experiments, male donor mice were primed by intravenous injection with 5-fluorouracil (5-FU; 200 mg/kg) 4 d before harvest. Male donors were killed by CO2 asphyxiation, femur and tibia were collected, and bone marrow was harvested by flushing with syringe and 26-gauge needle. Cells were counted and plated without removal of erythrocytes at 2 × 107 cells per 10-cm plate in prestimulation medium (38 (link)) of DME, 15% (vol/vol) inactivated FCS, 5% (vol/vol) WEHI-3B conditioned medium, penicillin/ streptomycin, 1.0 μg/ml ciprofloxacin, 200 μM l-glutamine, 6 ng/ml recombinant murine IL-3 (Genzyme), 10 ng/ml recombinant murine IL-6 (Genzyme), and 50–100 ng/ml recombinant murine stem cell factor (SCF; PeproTech). With non–5-FU–treated marrow, 10 ng/ml recombinant murine IL-7 (Genzyme) was also included. After prestimulation for 24 h at 37°C, viable cells were counted and transduced with retroviral stocks in the same medium containing 50% retroviral supernatant, 10 mM Hepes, pH 7.4, and 2 μg/ml polybrene. To increase transduction efficiency (39 (link)), virus and cells were cosedimented at 1,000 g for 90 min in a Sorvall RT-6000 centrifuge. Medium was changed after a 2–4-h adsorption period. At 48 h, a second round of transduction and cosedimentation was performed, and the cells were collected 2 h later, washed once in HBSS, and counted. Recipient female mice were prepared by two doses of 450-cGy gamma irradiation separated by 3 h. Transduced marrow cells were transplanted by injection of 0.2–0.5 × 106 cells (5-FU–treated marrow) or 1.0 × 106 cells (non–5-FU–treated marrow) in 0.4–0.5 ml HBSS into the lateral tail vein. After transplant, recipients were housed in microisolator cages supplied with acidified (pH 2.0) water.