Figure 1

Insertional mutation of murine Topors. A. Schematic of the gene-trap insertion into intron 2 of Topors. The three Topors exons are shown as rectangles. In exon 3 R represents the conserved RING domain required for ubiquitination activity, and S represents the region involved in sumoylation activity. The SA (splice acceptor), β-geo (β-galactosidase-neomycin) fusion gene, and pA (polyadenylation) sequences present in the gene-trap vector are shown. Locations of PCR primers used for genotyping are indicated by arrows. B. Representative PCR-based genotyping of mice obtained from breeding heterozygotes. C. Analysis of Topors mRNA and protein expression in colon tissue obtained from Topors^+/+, Topors^+/-, and Topors^-/- mice. As indicated in the Methods, RNA expression was evaluated by RT-PCR using primers spanning exons 2-3 and primers within exon 3 only. Actin RNA expression was determined using β-actin-specific primers. Protein expression was evaluated by immunoblotting nuclear lysates with a polyclonal antibody generated against the human protein, as well as a β-actin antibody. D. Kaplan-Meier survival analysis of Topors^+/+ (n = 40), Topors^+/- (n = 69), and Topors^-/- (n = 7) mice that survived weaning. The insert shows spinal kyphosis in a Topors^-/- mouse that was sacrificed at 68 weeks of age.