(A) CD45.2 BM cells isolated from either Prmt5^fl/fl, Mx1Cre⁺ Prmt5^fl/+, or Mx1Cre⁺ Prmt5^fl/fl mice were transplanted into recipient mice, together with WT CD45.1 BM cells. The ratio of CD45.2 to CD45.1 in peripheral blood mononuclear cells and the percentages of CD45.2 cells in BM, spleen, and thymus were determined by FACS analysis 7, 14, and 21 d.p.i. (n ≥4). (B) The frequency of CD45.2⁺ LK and LSK cells in BM from transplanted mice was determined by FACS analysis 7 and 14 d.p.i. (n = 5). (C) The absolute number of CD45.2 LT-HSCs (CD48^–CD150⁺ LSK cells) in the transplanted mice 14 d.p.i. was calculated and plotted as the mean ± SD (n = 4). (D) The percentage of Ki67⁺ LT-HSCs in CD45.2 and CD45.1 BM cells was determined by FACS analysis. Results are representative of 2 independent transplantation experiments, with 3 to 4 mice per group in each experiment. (E) CFU assays were performed using day-7 BM cells transduced with either empty lentivirus (pBGJR) or viruses expressing PRMT5wt or PRMT5EQ proteins. GFP⁺ cells were flow sorted and plated in methylcellulose cultures in duplicate. Colonies were scored 7 days after plating. Results are representative of 3 independent experiments using cells from 2 mice of the same genotype pulled together for viral transduction. (F) CAFC assays were performed using day-5 BM cells isolated from Prmt5^fl/fl or ERCre⁺ Prmt5^fl/fl mice. Cells were transduced with pBGJR or PRMT5wt-, or PRMT5EQ-expressing lentiviruses and were cocultured with MS5 cells in the presence of 0.1 μM 4-OHT. The number of cobblestone areas at the end of the coculture is plotted as the mean ± SD (n = 3). All P values were determined by a 2-tailed Student’s t test.