Transplantation of Gfi1b-deficient, flow-sorted HSCs. (A) Fifty HSCs originating from either wt (CD45.1) or Gfi1b^ko/ko (CD45.2) mice were transplanted into lethally irradiated CD45.1⁺ mice. Twenty-four weeks after transplantation, mice were euthanized and examined for the contribution of Gfi1b-deficient HSCs to the different lineages. (B) Percentage of CD45.2–positive cells (%CD45.2) in the blood at indicated time points after transplantation (n = 3). (C) CD45 chimerism in the blood was determined 24 weeks after transplantation in recipient mice (n = 3) overall (All) and for the indicated lineages. Myeloid (Mac-1), B-lymphoid (B220), T-lymphoid (CD3). The difference is significant (P ≤ .05) for CD45 chimerism between wt and Gfi1b-deficient cells, when all leukocytes are taken into account (All). (D) CD45 chimerism was determined 24 weeks after transplantation in the blood, bone marrow, spleen, and thymus of recipient mice (n = 3). (E) The frequency of bone marrow HSCs in mice either transplanted with wt CD45.1 and wt CD45.2 HSC cells (white) or mice transplanted with wt CD45.1 and Gfi1b-deficient (MxCre tg Gfi1b^fl/fl) HSCs (black) was determined (n = 3, P ≤ .01). (F) Quantification which proportion of HSCs originates from CD45.2 wt or CD45.2 Gfi1b-deficient HSCs in mice transplanted with either wt CD45.1 and wt CD45.2 HSCs or mice transplanted with sorted HSC cells from wt CD45.1 and Gfi1b-deficient CD45.2 mice (MxCre tg Gfi1b^fl/fl) (n = 3, P ≤ .01). (G) Frequency of HSCs circulating in the peripheral blood of mice either transplanted with wt CD45.1 and wt CD45.2 HSCs or mice transplanted with wt CD45.1 and Gfi1b-deficient CD45.2 HSCs (n = 3, P ≤ .01). (H) Quantification of which proportion of HSCs circulating in blood originates from CD45.2 wt or CD 45.2 Gfi1b-deficient HSCs in CD45.1 mice transplanted with wt CD45.1 and wt CD45.2 HSCs or CD45.1 mice transplanted with wt CD45.1 and Gfi1b-deficient HSCs (n = 3, P ≤ .01). (I) Quantification of which proportion of Lin⁻, Sca-1⁺, c-kit⁺ (LSK) cells in bone marrow originate from CD45.2 wt or CD 45.2 Gfi1b-deficient HSCs in mice transplanted with wt CD45.1 and wt CD45.2 HSCs or CD45.1 mice transplanted with wt CD45.1 and Gfi1b-deficient HSCs (n = 3, P ≤ .01). (J) Serial transplantation. Mice were transplanted with bone marrow from wt CD45.1 and Gfi1b-deficient (CD45.2) mice. After 24 weeks, chimerism in peripheral blood was determined, and 2 million bone marrow of these chimeric mice was transplanted into new lethally irradiated CD45.1 recipient mice. After 16 weeks, chimerism in the blood in these secondary transplanted mice was determined. The percentage of CD45.2 cells in the blood of the secondary transplant recipients was compared with the percentage of CD45.2 cells from the first transplant. The observed chimerism in the first transplant was set to 100% (n = 7 for second transplant, P ≤ .15). (K) Cells from 50 μL of blood were obtained from wt CD45.2 or Gfi1b-deficient CD45.2 mice and were transplanted together with 200 000 bone marrow cells from wt CD45.1 mice. Twelve weeks after transplantation, number of CD45.2 cells (which was set to 1 for CD45.2 Gfi1b-deficient blood cells) within all hematopoietic cells (CD45) in blood was determined. As a control for specificity of the CD45.2 antibody, blood obtained from an untreated CD45.1 mouse was used.