Targeted disruption of the synoviolin/hrd1 gene. (A) Structures of the synoviolin/hrd1 wild-type allele, the targeting vector, targeted allele, and the partial restriction map of the genes before and after targeting events. Exons of the gene are shown as closed boxes, and the β-galactosidase gene (LacZ), neomycin phosphotransferase gene (Neo), diphtheria toxin-A gene (DT), and pBluescript II (BSK) are shown as open boxes. The restriction sites used are as follows: (B) BglII; (P) PstI; (E) EcoRI; (X) XhoI; and (N) NcoI. (B) Southern blot analysis of genomic DNA extracted from E13.0 embryos generated by intercrosses of syno+/-. The DNA were digested with PstI (left) or BglII (right) and probed with an external probe. (C) Northern blot analysis. Twenty micrograms of total RNA, isolated from E13.0 embryos generated by intercrosses of syno+/-, hybridized with the probe for synoviolin/hrd1 or G3PDH probe. (D) The E13.0 embryos total protein was isolated and separated by SDS-PAGE (50 μg protein/lane). After transfer of the protein, the membrane was probed with anti-Synoviolin/Hrd1 antibody.