Generation of Hoxb4-deficient mice. (A) The targeting construct compared with the WT locus. Gray arrowheads indicate loxP sites; black boxes, Hoxb4 exons. (B) Targeted locus showing the strategy for detection of homologous recombination (HR) events. Black arrows indicate PCR primers (P1-P5), and resultant bands are shown in panel D. Two Southern blot strategies are shown. The PstI digest using the 3′ external probe confirms the homologous integration of the 3′ loxP site, and the KpnI digest using the neo probe verifies a single homologous integration site for the construct (see panels E and F). (C) Targeted locus after Cre-mediated recombination events, resulting in either a floxed or a deleted Hoxb4 gene. (D) Findings from the PCR analysis verifying homologous recombination at the 5′ end of the construct (P1 and P2) and the presence of the 3′ loxP site (P3 and P4). (E-F) Southern blot analysis using KpnIor Pst I digests, respectively. All the clones (3, 4, and 7) have a single integration site of the neomycin cassette, but clone 4 does not have the single 3′ loxP site. (G) RT-PCR results showing normal expression of Hoxb4 in floxed mice, but no expression can be detected in mice carrying the deleted version. (H) PCR analysis for genotyping mice that carry the deleted Hoxb4 version. Neo indicates neomycin-resistance gene driven by the thymidine kinase promoter; PGK-tk, thymidine kinase driven by the PGK promoter; HdIII, HindIII; ERI, EcoRI; ext probe, external probe; and kb, kilobase pairs.