Figure 2.

Targeted disruption of the Sycp2 gene. (A) Schematic diagram of the Sycp2 targeting strategy. Neomycin selection marker (PGK-Neo) was flanked by loxP sites. Thymidine kinase (TK) was included for negative selection with ganciclovir. Exons are shown in black boxes and designated by the numbers shown above the boxes. (B) Splicing of the mutant Sycp2 transcript. RT-PCR was done with poly(A)⁺ RNAs from wild-type (+/+) and Sycp2 mutant (−/−) testes. PCR primers are located in exons 38 and 44. PCR product size is shown in bps. (C) Production of the mutant SYCP2t protein. Western blot analysis was performed on wild-type, Sycp2 ^+/−, and Sycp2 ^−/− testicular protein extracts using rabbit anti-SYCP2 serum. (D) In vivo association of SYCP3 with SYCP2, but not SYCP2t. Soluble nuclear protein extracts from wild-type and Sycp2 ^−/− testes were immunoprecipitated with anti-SYCP3 antibodies. Immunoblotting was probed with anti-SYCP2 antibodies. SYCP2, but not SYCP2t, was coimmunoprecipitated with SYCP3. *, a nonspecific diffuse band observed in the immunoprecipitated (IP) pellet of both wild-type and mutant. IP, immunoprecipitation; NE, nuclear extract; Sup, supernatant. Protein molecular mass standards are shown in kilodaltons.