American Society of Hematology

Figure 2.

$Figure 2. Stem cells from pIpC-treated Runx1F/F—Tg(Mx1-Cre) mice are reduced in competitive repopulation ability. (A) Ethidium bromide (EtBr)–stained 3% agarose gel of 3-primer PCR of Runx1 loci from sorted HSCs derived from fresh BM 17 weeks after pIpC (Table 1) and unfractionated BM and PB. 1 = Runx1F/F; 2 = RunxF/F—Tg(Mx1-Cre). Mr = 1-kb ladder, sizes indicated (bp). Control samples include tail DNA from Runx1+/+, Runx1F/+, and Runx1F/F mice and from Runx1Δ/Δ, Runx1F/Δ, and Runx1+/Δ embryos. Runx1Δ alleles were generated by mating Runx1F/F mice to Tg(EIIa-Cre) mice and intercrossing Runx1F/Δ mice. (B) Composite showing gating strategy and CD45.1/CD45.2 staining of PB from representative mice that underwent transplantation with Runx1F/F or Runx1F/F—Tg(Mx1-Cre) and competitor marrow 8 weeks after transplantation. Green indicates lymphocytic (R2), and red indicates granulocytic (R3) fractions based on scatter gating (SSC, side scatter; FSC, forward scatter). Gate assignments were confirmed by back-gating of control stainings with Gr1+–, Mac1+–, CD3+–, T-cell receptor β (TCRβ+)–, CD19+–, or B220+–stained PB (not shown). The percentage contribution to the granulocytic and lymphocytic lineages of PB from each marrow isotype (CD45.1/CD45.2) was determined. (C) Plotted is the mean (±SD) percentage contribution of donor-derived CD45.2+ cells in PB from mice that underwent transplantation with Runx1F/F—Tg(Mx1-Cre) (red symbols; n = 6) or Runx1F/F (black symbols; n = 6) and competitor bone marrow to total WBCs, granulocytes, and lymphocytes of recipient mice at 4, 8, and 19 weeks after transplantation. Two of 8 mice receiving Runx1F/F—Tg(Mx1-Cre) marrow that failed to show contribution of CD45.2+ marrow to PB of recipient mice at any time point are excluded. (D) Nineteen weeks after transplantation, EtBr-stained 3% agarose gel of 3-primer PCR of Runx1 loci of BM from mice that underwent competitive transplantation. (E) EtBr-stained 3% agarose gel of 3-primer PCR of Runx1 loci from BM of mice that underwent transplantation without competitor marrow.$

Stem cells from pIpC-treated Runx1^F/F—Tg(Mx1-Cre) mice are reduced in competitive repopulation ability. (A) Ethidium bromide (EtBr)–stained 3% agarose gel of 3-primer PCR of Runx1 loci from sorted HSCs derived from fresh BM 17 weeks after pIpC (Table 1) and unfractionated BM and PB. 1 = Runx1^F/F; 2 = Runx^F/F—Tg(Mx1-Cre). Mr = 1-kb ladder, sizes indicated (bp). Control samples include tail DNA from Runx1^+/+, Runx1^F/+, and Runx1^F/F mice and from Runx1^Δ/Δ, Runx1^F/Δ, and Runx1^+/Δ embryos. Runx1^Δ alleles were generated by mating Runx1^F/F mice to Tg(EIIa-Cre) mice and intercrossing Runx1^F/Δ mice. (B) Composite showing gating strategy and CD45.1/CD45.2 staining of PB from representative mice that underwent transplantation with Runx1^F/F or Runx1^F/F—Tg(Mx1-Cre) and competitor marrow 8 weeks after transplantation. Green indicates lymphocytic (R2), and red indicates granulocytic (R3) fractions based on scatter gating (SSC, side scatter; FSC, forward scatter). Gate assignments were confirmed by back-gating of control stainings with Gr1⁺–, Mac1⁺–, CD3⁺–, T-cell receptor β (TCRβ⁺)–, CD19⁺–, or B220⁺–stained PB (not shown). The percentage contribution to the granulocytic and lymphocytic lineages of PB from each marrow isotype (CD45.1/CD45.2) was determined. (C) Plotted is the mean (±SD) percentage contribution of donor-derived CD45.2⁺ cells in PB from mice that underwent transplantation with Runx1^F/F—Tg(Mx1-Cre) (red symbols; n = 6) or Runx1^F/F (black symbols; n = 6) and competitor bone marrow to total WBCs, granulocytes, and lymphocytes of recipient mice at 4, 8, and 19 weeks after transplantation. Two of 8 mice receiving Runx1^F/F—Tg(Mx1-Cre) marrow that failed to show contribution of CD45.2⁺ marrow to PB of recipient mice at any time point are excluded. (D) Nineteen weeks after transplantation, EtBr-stained 3% agarose gel of 3-primer PCR of Runx1 loci of BM from mice that underwent competitive transplantation. (E) EtBr-stained 3% agarose gel of 3-primer PCR of Runx1 loci from BM of mice that underwent transplantation without competitor marrow.

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