The C-terminal 95 amino acids of CBFβ-SMMHC are required for defects in hematopoiesis and leukemogenesis. (A) Targeting strategy used to replace exon 5 of Cbfb with the targeting construct. Locations of probe 0.2C and the sizes of NcoI fragments detected by 0.2C are indicated. Filled triangles represent lox-P sites. (B) Southern blot hybridization of NcoI-digested DNA from transfected ES cell clones with probe 0.2C. The 15.7 kb band corresponds to the wildtype Cbfb allele, and the 6.8 kb band corresponds to the knockin allele. (C) Western blot analysis of parental (Cbfb^+/+), full-length CBFβ-SMMHC (Cbfb^+/MYH11) expressing, and CBFβ-SMMHCΔC95 (Cbfb^+/ΔC95) expressing ES cell clones. The blot was probed with an antibody specific to CBFβ. The calculated molecular weights for each protein are indicated. (D) Representative FACS plots for Ter119, C-Kit, and Csf2rb staining of primitive blood from E10.5 embryos from line No. 1 of the indicated genotypes. Percentage of cells in each gate is given. N ≥ 3 for each genotype. (E) Histologic sections of fetal livers from E12.5 embryos from line No. 2 of the indicated genotypes. Yellow arrows indicate megakaryocytes. White arrows indicate hematopoietic progenitors. Similar results were found in embryos from line no. 1 (data not shown). (F) Representative FACS plots of B220, C-Kit, Gr1, and Mac1 of peripheral blood from older than 10-month-old mice from line no. 1 of the indicated genotypes. Percentage of cells in each gate is given. N > 3 for each genotype. (G) Wright-Giemsa stained peripheral blood smears from older than 10-month-old mice of the indicated genotypes from line no. 1. Similar results were observed in mice from line no. 2 (data not shown).