Figure 1.

Illustration of the experimental protocol used to investigate the competitive repopulating ability (CRA) of mouse HSCs transduced with lentiviral vectors related to HSCs not exposed to lentiviral particles. Prestimulated Lin⁻Sca1⁺ BM cells from male Ly5.1 mice were exposed to LVs, as described in “Transduction of Lin⁻Sca1⁺ cells.” Another Lin⁻Sca1⁺ population from male Ly5.2 mice was treated under identical conditions without LVs. After a total incubation period of 48 hours, cells were mixed 1:1 and then transplanted into irradiated recipients. Nine months after transplantation, BM from primary recipients was retransplanted into secondary recipients. At periodic intervals, peripheral blood cells from primary and secondary recipients were sampled to determine the proportion of Ly5.1⁺ and EGFP⁺ cells (Figures 2,3). In one experiment, BM from secondary recipients was retransplanted into tertiary and quaternary recipients to conduct further studies of hematopoietic repopulation (Figure 4). In this experiment, BM cells from primary and quaternary recipients were analyzed by LAM-PCR to determine the LV integration sites in the corresponding repopulating cells (Figure 5).