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. Author manuscript; available in PMC: 2020 Feb 4.
Published in final edited form as: Neurobiol Dis. 2019 Nov 26;134:104696. doi: 10.1016/j.nbd.2019.104696

Fig. 1.

Fig. 1.

Cdnf knockout mice are viable, fertile and show no obvious gross phenotypes. (A) Schematic illustration of the WT Cdnf+/+ and targeted CdnfPu/Pu and Cdnf−/− loci. Arrowheads indicate priming sites used in genotyping by PCR and in RT-PCR. A PGK-promoter driven puromycin selection cassette (PuroΔtk), exon (E), Frt (F)-site, LoxP (L)-site. (B). Example of genotyping result. Genotyping of mice was performed by PCR using primers forward 1 (f1), forward 2 (f2) and forward 3 (f3) and reverse 2 (r2), indicated by arrowheads in (A). Amplified band of 201 bp represents WT Cdnf+/+, band of 300 bp represents the targeted CdnfPu/Pu allele, where puromycin cassette is included (left panel). In the right panel, amplified band of 400 bp represents the targeted allele from which the puromycin cassette has been deleted, and the 271 bp band the WT band. MWM; molecular weight marker. (C) RT-PCR analysis for Cdnf and Manf mRNA expression in tissues from adult Cdnf+/+ and Cdnf−/− mice. Primers for Gapdh were used to normalize mRNA levels in each sample. (D) Western blot analysis of tissue-lysates confirms that CDNF protein is not expressed in tissues of Cdnf−/− mice. Recombinant human CDNF protein (rhCDNF) was used as a positive control and anti-actin antibody for normalization of total protein content. (E) Growth curve of Cdnf+/+ (n = 5) and Cdnf−/− (n = 5) littermate male mice. Body weight was measured weekly from postnatal day (P) P3 to 11 weeks of age (two-way RM ANOVA, interaction of genotype and time, F(11,88) = 1.08, p = .387). Weight of 1 year-old Cdnf+/+ and Cdnf−/− female and male mice in (F) C57BL/6 background (n = 6–14, two-tailed t-test), and (G) ICR/C57BL/6 mixed background (n = 6, two-tailed t-test, males; Mann Whitney U test). (H) Analysis of the indicated components from blood serum of 1 year-old Cdnf+/+ (n = 12) and Cdnf−/− (n = 14) male mice. Differences were analyzed with t-test and calcium content by t-test with Welch’s correction. (I) Ad libitum fed blood glucose levels from 1 year-old Cdnf+/+ and Cdnf−/− mice in C57BL/6 background (n = 19–20, males; t-test (Welch’s correction) and mixed background (n = 9–10, males; t-test). (J) Organ weights normalized to body weight did not differ between genotypes in 2 year-old (males, n = 4, 5) Cdnf+/+ and Cdnf−/− mice (t-test; for brain, liver, gut, testicles and spleen, Mann Whitney U test). Mean ± SEM. F, female, M, male.