Figure 1.
Generation and characterization of Clcn5 knockout mice
(A) Gene structure of mouse Clcn5 and the sgRNAs used for deleting the 26 kbp region. The two dashed red boxes indicate the replaced regions in the two published gene knockout strains (Piwon and co-workers26,27). The asterisk indicates the position of the point mutation in the published point muation mouse model (Novarino et al.7). (B) RT-PCR confirming the lack of Clcn5 mRNA expression in the kidneys of mutant mice. Two wild-type and two mutant mice (2 months old) were analyzed. “RT-”: RNA from the kidney of a wild-type mouse was directly used as a PCR template without reverse transcription. Primers were specific to mouse Clcn5 cDNA. (C) Western blotting confirming the lack of ClC-5 protein in the kidney tissues of mutant mice. (D) Volume, calcium, and protein in urine of female mice. Wild-type, heterozygous, and homozygous mutant mice were 81 days old. ∗p < 0.05 between the indicated groups (Dunn's multiple comparison test following Kruskal-Wallis test). (E) Volume, calcium, and protein in urine of male mice. Urine samples were collected from 2- to 2.5-month-old mice. ∗∗∗p < 0.0001 between wild-type and mutant mice (Mann-Whitney test).