Fig. 1. Generation of an oocyte-specific GCNF knockout mouse model. (A) Structures of the GCNF^3lox, GCNF^fl and GCNF^lox alleles and the targeting vector. LoxP sites, filled triangles; the DBD-encoding exon, filled box. Restriction enzyme sites of Acc65I (Ac), ApaI (Ap), BglI (Bg), BsiEI (Bs), EcoRV (Rv), XhoI (X) and SalI (S) in each allele are shown. Positions of PCR primers (P1 to P6), and the 5′ probe for Southern blot analysis are indicated. (B) Cre recombinase activity on the floxed ROSA transgene in the ovaries determined by β-galactosidase staining. Bar scale, 200 µm. (C) Genotype analysis of tail biopsies. (a) Southern blot analysis showing a 20 kb band for the GCNF⁺ allele and a 10 kb band for the GCNF^fl allele. (b) PCR analysis using primers (P3 and P4) to distinguish the GCNF^fl and GCNF⁺ alleles, and primers (Cre-A and Cre-B) to determine the presence of Zp3Cre transgene. (D) RT–PCR analysis showing the loss of the DBD encoding region of the GCNF mRNA in the GCNF^fl/flZp3Cre⁺ ovary. (E) Genotyping of tail DNA showing the complete deletion of the GCNF DBD encoding exon in the progenies from the cross between GCNF^fl/flZp3Cre⁺ females and wild-type males. (F) PCR analysis showing complete deletion of the GCNF DBD encoding exon in ovulated oocytes, but not in cumulus cells. (G) In situ hybridization showing the loss of the DBD region of the GCNF mRNA in the oocytes of GCNF^fl/flZp3Cre⁺ mice. Bar scale, 50 µm.