Experimental procedure and measurements of chimerism.

(A) Schematic representation of the HSC transplantation protocol. Bone marrow cells were isolated from donor mice injected 3 days previously with 5-fluorouracil (5-FU) and were cultured for 48 hours in medium containing stem-cell factor, IL-3, and IL-6 to stimulate proliferation of HSCs. Cells were subsequently transduced for 48 hours, using ecotropic retroviral supernatants and culture dishes coated with fibronectin. The transduction procedure incorporated replacement of the retroviral supernatant after 24 hours and 2 rounds of centrifugation for 2 hours to increase the effective viral titer. After transduction, cells were harvested and injected intravenously into recipient mice that had been lethally irradiated (10.5 Gy) 24 hours previously. (B) Levels of chimerism were measured by FACS analysis of thioglycollate-elicited cells from Ly-5.1 recipient mice that had received transplants of Ly-5.2 HSCs. Cells were stained with biotinylated antibodies recognizing Ly-5.1 or Ly-5.2 and PE-conjugated streptavidin. The percentage of PE-positive cells was determined by use of a gate set to exclude unstained control cells. Data for each RV are from a single mouse but are representative of results from 5 other mice.