XbaI; β-gal, β-galactosidase; Neo, neomycin-resistant cassette; TK, thymidine kinase. b, c, Genotyping of mice by Southern blot using tail DNA. Genomic DNA was digested with NcoI and hybridized with 5 flanking probe (b) or doubly digested with EcoRV plus SacI and hybridized with 3 flanking probe (c). KO, knockout allele; WT, wild-type allele; p–/m+, paternal-deficiency heterozygotes. d, Genotyping of mice from heterozygous breeding. e, Northern-blot analysis of Ndn expression in male (M) and female (F) mice in brain (B) and liver (L) of wild-type (+/+) and paternal-deficiency heterozygotes (p–/m+). The probe was a 2.3-kb BamHI− HindIII fragment covering the ORF and 3 UTR of Ndn. f, Northern blot of the same filter as in (e) analysed with Gapd as a control. g, Growth curves of mice of various genotypes. Filled square, wild type (n= 10); diamond, paternal-deficiency heterozygotes (m+/p−, n= 10); circle, maternal-deficiency heterozygotes (n= 6); filled triangle, homozygotes (n= 8). The filled triangles are not visible and for the most part overlap the filled squares, indicating the similarity between wild-type and homozygous mice. The deletion was introduced into the AB2. 2 ES cell line and Southern-and northern-blot analyses were performed as described13. a b c d e f g

3 probe 5 probe

5 probe p–/m+ p–/m+