Competitive transplantation experiment shows that Dot1l is required for functional HSC activity. CD45.2⁺Dot1l^wt/wt;CreER⁺ (Dot1l^+/+) or Dot1l^F/F;CreER⁺ (Dot1l^F/F) mice were injected with tamoxifen and 3 days later bone marrow cells were mixed at 1:1 or 3:1 ratio with CD45.1⁺ competitors for transplantation. Recipients were lethally irradiated (900 cGy) CD45.1⁺ congenic mice. (A) Bar graph of CD45.2⁺ cells in peripheral blood. Peripheral blood was stained for myeloid (Gr1⁺, CD11b⁺), B lymphoid (B220⁺, CD19⁺), and T lymphoid (CD3⁺) cells. Blood was collected from recipients on indicated weeks after transplantation. Dot1l^F/F cells minimally constituted recipient peripheral blood. Data are mean ± SD. (B) Bar graph of CD45.2⁺ cells in recipient bone marrow and thymus 16 weeks after transplantation. Cells were stained for HSCs (lineage⁻, sca1⁺, ckit⁺, CD48⁻, CD150⁺), myeloid (Gr1⁺, CD11b⁺), B lymphoid (B220⁺, CD19⁺), and developing T cells (CD4⁺, CD8⁺). Even at 3:1 ratio, Dot1l^F/F cells failed to reconstitute recipient hematopoietic system. Data are mean ± SD. (C) Staining profile of recipient HSCs (lineage⁻, sca1⁺, ckit⁺, CD48⁻, CD150⁺) 16 weeks after transplantation. Percentages are based on HSC gate.