Smc3 is required for HSC function. (A) Schematic depiction of the targeted Smc3 allele. Exon 4 is targeted and flanked by LoxP sites upon Frt-mediated deletion of the Neo cassette. (B) Qiaxel gel electrophoresis image of PCR genotyping of the Smc3 WT allele (287 bp), Smc3 floxed allele (313 bp), and Cre recombination allele (349 bp). The genotype is shown for Smc3^+/+, Smc3^fl/+, Smc3^Δ/+, Smc3^fl/fl, and Smc3^Δ/Δ. (C) Peripheral blood WBCs, hemoglobin (Hgb), and platelets after postnatal deletion of Smc3. Counts are compared with age-matched controls injected with PIpC (n = 6 for each genotype). (D) Kaplan-Meier curve of primary mice after postnatal Smc3 deletion (n = 10 for each genotype). (E) Histological (H&E) analysis of Mx1-Cre Smc3^Δ/Δ and Cre-negative Smc3^fl/fl control BM. (F) Nucleolar stain of Mx1-Cre Smc3^Δ/Δ BM reveals fragmented and supernumerary nucleoli. ACK-lysed BM was stained with TOTAL-NUCLEAR-ID fluorescent reagents, allowing simultaneous staining of both the nucleoli (green) and total nucleus (red). (G) Flow cytometric enumeration of B220⁺, Cd11b⁺/Gr1⁺, Cd3⁺, and Cd4/8 ratio of cells in the peripheral blood of Mx1-Cre Smc3^fl/fl mice and cre-negative controls. (H) Metaphase karyotyping upon exposure to colcemid (45 min): Representative metaphases with partial or complete premature sister chromatid separation resulting in tetraploidy/polyploidy in 44/200 cells (22%). (I and J) Competitive transplantation of 500K donor-derived Cd45.1 whole BM was injected by tail vein after lethal irradiation with either 500K Cd45.2 whole BM from Mx⁻ Smc3^fl/fl or 500K Mx⁺ Smc3^Δ/Δ (n = 5 mice each genotype). Mice were treated with PIpC at week 2, and chimerism was measured by percentage of Cd45.2 in the peripheral blood every 4 wk. Error bars represent ± SD (C, I, and J); ***, P < 0.001 (Mann–Whitney U test).