Generation of Trip13 Mutant Mice
To explore the function of TRIP13 in mammals, we generated mice with a gene trap-disrupted allele, Trip13RRB047 (Figure 1C; abbreviated as Trip13Gt). Heterozygotes were normal in all respects, but homozygotes were present at ∼2/3 the expected ratio from intercrosses between heterozygotes (91 Trip13 +/+, 183 Trip13Gt/+, and 61 Trip13Gt/Gt). Since >90% of prewean mice that died were mutant homozygotes, this discrepancy is apparently due to a partially penetrant lethality. Most surviving Trip13Gt/Gt animals were grossly normal. However, homozygotes that were semi-congenic (N4) on the C57BL/6J strain were often markedly smaller and/or had kinked or shorter tails (Figure 2A and 2B).
Figure 2  Developmental Phenotypes of Trip13 Mutant Mice
(A) Shown are 21-d-old littermates. Note the shortened tail in the mutant, but overall similar body size.
(B) Shown are 23-d-old littermates. The mutant is smaller in this case, but the tail is not as truncated as the mouse in (A).
(C) Wild-type (WT) and homozygous Trip13 mutant (MUT) testes.
(D) and (E) are cross sections through 17.5-d-old heterozygous (“WT”) and homozygous mutant Trip13 testes, respectively. Whereas the tubules in WT show coordinated spermatogenesis with pachytene spermatocytes present in all tubules (proximal to the lumen), developmental progression in the mutant is not synchronized between tubules. Some tubules have no pachytene spermatocyes (asterisks), while in others, development is somewhat disorganized (#). RT-PCR analysis of Trip13Gt expression (Figure 1D) revealed a low level of normally spliced transcripts in testes of homozygotes that is presumably a consequence of incomplete usage of the gene trap's splice acceptor. Western blot analysis, using a polyclonal antibody raised against a peptide encoded by exon 3, revealed multiple species in wild-type and heterozygous testes, one of which corresponds to the expected size of 48 kDa (Figure 1E). This and three other species were undetectable in homozygous mutant testes, but a reduced amount of an intense ∼38 kDa smaller band was present. It is not clear if this corresponds to TRIP13. The greatly decreased Trip13 mRNA and predicted correct-length protein in mutants indicate that the Trip13RRB047 allele is severely hypomorphic.
To determine the germ cell types in which TRIP13 is expressed, and to assess possible expression in the mutant by means other than Western analysis, testis sections were immunolabeled for TRIP13 using a polyclonal chicken antipeptide antibody (see Materials and Methods). The most intensely labeled cells in control testes were Type B spermatogonia and leptotene spermatocytes (Figure 1F). Zygotene/pachytene spermatocytes stained less strongly, and there was no detectable staining in late pachytene spermatocytes. TRIP13 appeared to be nuclear localized. There was no such staining of nuclei in mutant seminiferous tubules (Figure 1F). To further assess the nuclear localization, TRIP13 was used to probe meiotic chromosomes prepared by surface spreading of spermatocyte nuclei. In wild type, there was diffuse nuclear staining, and no evidence of concentration on SC cores (marked by the axial element protein SYCP3) at any meiotic substage (Figure 1G). TRIP13 signal was noticeably absent in mutant meiotic nuclei.