Fig. 3

Generation of Runx1 ^P2TAA mutant mice. a Schematic representation of the targeting strategy used to generate the Runx1 ^P2TAA allele. A targeting vector was designed to replace ATG with TAA, marked with *. Open and closed boxes represent the 5′ untranslated region (UTR) and coding region in exon II, respectively. Neomycin resistance (neo ^r) and thymidine kinase (tk) genes were used for positive and negative selection, respectively. Triangles represent loxP sequences. b Sequence analysis of the genomic region around the ATG in exon II of the Runx1 gene in an ES clone showing replacement of ATG with TAA. c Gel image of DNA-PCR analysis showing incorporation of the P2-promoter deletion in an ES clone. d One representative dot plots of at least three individual experiments showing ScaI and c-Kit expression in Lin-negative (Lin⁻) fetal liver cells from 11.5 dpc Runx1 ^+/+ and Runx1 ^P2TAA/P2TAA embryos. e One representative of two colony forming assay of fetal liver cells from mice of indicated genotypes. f Genotyping of offspring obtained by intercrossing Runx1 ^+/P2TAA heterozygous mice at 15.5 dpc and four weeks (P28). Numbers and those in parenthesis represent live and total mice, respectively. g Dot plots showing CD4 and CD8 expression in total thymocytes of three week-old Runx1 ^+/+ and Runx1 ^P2TAA/P2TAA mice. h Graph showing absolute numbers of thymocyte subsets. DN: CD4⁻CD8⁻ double negative. DP: CD4⁺CD8⁺ double positive. CD4SP: CD4⁺CD8⁻ single positive. CD8SP: CD4⁻CD8⁺ single positive. Mean ± SD. ** P < 0.01. i Dot plots showing CD4 and CD8 expression in splenic T cells of three week-old Runx1 ^+/+ and Runx1 ^P2TAA/P2TAA mice. j Graph showing CD4⁺ to CD8⁺ T cells in lymph nodes. Mean ± SD. ** P < 0.01. k Immunoblot showing expression of Runx1 protein in total thymocytes from three week-old Runx1 ^+/+ and Runx1 ^P2TAA/P2TAA mice. One representative image of two experiments