FIG. 1.

Disruption of Siah2 by gene targeting. (A) Exons I, II, III, and IV of Siah2 are depicted as rectangles, the coding region of Siah2 is shown in black, and untranslated regions are shown in white. The targeting construct containing a TK gene cassette and a neomycin resistance gene cassette (NeoR) is depicted. DNA fragments used as the left and right arms of homology are represented by dashed lines. Homologous recombination yields the targeted locus in which the NeoR cassette is inserted into exon IV and introduces a new EcoRI restriction site. Abbreviations for restriction enzymes: B, BamHI; R, EcoRI. (B) Southern blot genotyping of progeny derived from an intercross of Siah2 heterozygous mice. Genomic DNA was digested with EcoRI and Southern blotted with a 1-kb probe located 3′ of the region of DNA used in the targeting construct (probe in panel A). The positions of the 13.2-kb wild-type (WT) and 3.7-kb knockout (KO) alleles are shown. The migration positions of known DNA molecular size markers are shown. (C) Northern blot analysis of 3 μg of poly(A)⁺ RNA isolated from brains of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) Siah2 mutant mice. A 410-bp ScaI-DraI fragment derived from the 3′-untranslated region of exon IV of Siah2 was used as a probe. Probing with a fragment from the glyceraldehyde-3-phosphate dehydrogenase gene served as a loading and transfer control. The migration positions of known RNA molecular size markers are shown. (D to G) Analysis of wild-type (D and E) and Siah2 mutant (F and G) ovaries by in situ hybridization using an antisense riboprobe derived from nucleotides 1159 to 1786 of the Siah2 cDNA. This probe includes the final 110 bp of the Siah2 coding region and extends into the 3′-untranslated region of exon IV. (D and F) Light-field illumination. Black triangles mark the positions of selected developing ovarian follicles. (E and G) Dark-field illumination. White triangles mark the positions of the selected developing ovarian follicles marked in panels D and F. Note the absence of Siah2 expression in ovaries from Siah2^−/− mice.