Fig. 1. Targeted disruption of mouse Par3 gene. (A) Schematic structures of the full-length PAR3 protein (180 kDa) and a major splice variant (100 kDa) expressed in mouse embryos. The third exon (E3) encodes amino acids 75-135. Anti-PAR3 antibodies were raised against amino acids 1-115 (N2-3E1) or 712-936 (C2-3AP). CR1 and PDZ indicate the conserved oligomerization and PDZ domains, respectively. (B) Restriction maps of wild-type mouse Par3 genomic locus, targeting vector, targeted allele (Par3^Neo) and Par3 deleted allele (Par3^E3) obtained after cre-mediated excision of loxP-flanked region, including the third exon (E3, 181 bp, boxes), which encodes amino acids 75-135. The targeting vector has one loxP-flanked MC1-neo cassette in intron 2, one loxP site in intron 3 and one MC1-DT-A-negative selection marker. The loxP sequences are shown in triangles. The position of the 5' external probe for Southern blot analysis is indicated. RV, EcoRV; Ap, ApaI; Nc, NcoI; Ns, NsiI; Nd, NdeI. (C) Southern blotting of EcoRV- or ApaI-digested genomic DNA from wild-type (+/+) and heterozygous (Neo/+) littermates. The size of each band yielded by the 5' external probe is indicated. (D) Southern blotting of EcoRV-digested genomic DNA from wild-type (+/+), heterozygous (+/E3) and homozygous (E3/E3) littermates. The 5' external probe yields an 8.2 kb band from the wild-type allele, and a 7.2 kb band from the Par3^E3 allele. (E) The loss of the PAR3 protein in embryos at E11.5 was examined by immunoblotting. Protein extracts from the heads of the embryos shown in D were probed with affinity-purified anti-PAR3 antibodies (C2-3AP, N2-3E1). An asterisk indicates nonspecific bands. The CBB staining of blotted membrane is shown as a loading control.