Analyses of Ap2a2-transduced HSCs In vitro. Cells used for panels A through E are CD150+48−Lin−. (A) CRU assay in primary recipients with respective transduced cells. (B) Southern blots with GFP-specific probe on BM of 11 recipient mice (2119-3491) from 4 independent culture wells of Ap2a2-transduced HSCs (1-4). (C) Donor-derived compared with competitor, recipient-derived CD150+48−LSK LT-HSCs in BM from primary recipients analyzed at 20 weeks after transplantation with vector- and Ap2a2-transduced HSCs. (D) MAS for transplanted Ap2a2-transduced HSCs as calculated from data in Figure 2B and C. Because these Ap2a2-transduced assays were performed in conjunction with our previous overexpression screen,20 in which the control CRU was known for freshly purified CD150+48−Lin− HSCs, the published data for fresh cells, days 0 and 7 grafts, and positive grafts for Hoxb4- and Trim27-transduced cells are used as valid negative controls and positive comparators, respectively. The MAS for Ap2a2 (*) is statistically significantly higher (see value in panel E), compared with day 0 and 7 controls. (E) P value for Ap2a2-transduced HSCs derived from MAS data in panel D compared with published control vectors (day 0 or 7) and the most potent HSC activators from our initial screen.20
