Runx1-IRES-GFP KI mice have decreased HSC pool. (A) GFP expression in HSPCs. GFP fluorescence in KI cells indicates Runx1 promoter activity. GFP histograms are shown by gating the indicated population. Dashed and solid lines denote WT and KI cells, respectively (2 mice each). (B) Flow cytometric analysis of adult BM. (C) Flow cytometric analysis of E14.5 fetal liver cells. (D) Competitive repopulation unit assay. The detailed protocol is described in supplemental Methods. WT cells have 7 times higher frequency of stem cells than KI cells. This experiment was repeated twice with similar results. (E) BMT of Runx1 overexpressing cells. One million cells with 20% GFP⁺ cells were injected into recipient mice. GFP percentage in peripheral blood was followed up. Ex6⁺, n = 4. Ex6⁻, n = 3. Ex6e, n = 4. Ex6⁺, Ex6⁻, and Ex6e represent Runx1bEx6⁺, Runx1bEx6⁻, and Runx1bEx6e, respectively. LT-HSC, long-term HSC.