Fig. 5. Disruption of the Ring1B gene in mice. (A) Diagram of the Ring1B locus, the targeting vector and of the modified allele. The Ring1B-coding exons are indicated by black boxes. The PGKneo and pMC1-tk expression cassettes were used for positive and negative selection, respectively. Position of relevant restriction sites (EcoRI, E; BamHI, B; HindIII, H; SalI, S; XhoI, X), location of probe and PCR primers, and sizes of diagnostic fragments are indicated. (B) Southern analysis of genomic DNA isolated from offspring of heterozygote matings after digestion with BamHI + HindIII and probed with the indicated 3' probe shown in A. (C) PCR analysis of tail genomic DNA of the offspring of a heterozygous intercross. Primers RB22 and RB18 amplify a 1.4 kb of the wild-type allele, whereas primers Neo2 and RB11 amplify a 3.0 kb of the mutated allele. (D) Semi-quantitative analysis of Ring1B by western blot analysis of total proteins extracted from 11.5 dpc mouse embryos of the indicated genotypes probed with antibodies against Ring1B. To correct for loading differences, anti-lamin B antibodies and Coomassie Brilliant Blue (CBB) staining of the same gel were used.

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