Figure 1

Construction of c-Myb-T572A knock-in mice. (a) The diagram of Fbw7-mediated regulation of T572-dependent mouse c-Myb. (b–f) Generation of mice carrying c-Myb-T572A. (b) The targeting construct encoding the murine c-Myb gene modified to express c-Myb-T572A (targeting vector) and schematic representations of the wild-type mouse c-Myb allele (WT); the recombinant c-Myb allele, which resulted from T572A amino acid exchange with a loxP-thymidine kinase (TK)-neomycin resistant (Neo) cassette [TA(neo)]; and the c-Myb-T572A knock-in allele after removal of the TK-Neo cassette by Cre recombinase (TA). Exons and loxP sites are shown as open boxes and triangles, respectively. The expected sizes of bands in a PCR analysis with primer set 1 (F1 and R1), primer set 2 (F2 and R2), and primer set 3 (F2 and R3) and a Southern blot analysis with probe [for SacI (Sc) fragments] are indicated. (c,d) Screening of homologous recombinant embryonic stem (ES) clone by PCR analysis (c) and Southern blot analysis (d). (c) Genomic DNA from ES clones were subjected to PCR with the primers F1 and R1. The position of the amplified fragment (0.95 kb) is indicated in (b). (d) Genomic DNA was digested with SacI and subjected to hybridization with probe. The wild-type and homologous recombinant alleles gave rise to hybridizing fragments of 4.8 and 3.0 kb, respectively. (e) Confirmation of a floxed ES clone. Genomic DNA from ES clones was analyzed by PCR with the primers F2 and R2. The positions of amplified fragments corresponding to wild-type (0.28 kb), homologous recombinant (5 kb) and knock-in (0.5 kb) alleles are indicated in (b). (f) Genotyping of c-Myb DNA extracted from F₇ mouse ears. The position of primers (F2, R2, and R3) used for the genotyping is indicated in (b).