Figure 1

Generation of Clr-f^−/− mice by targeted Clr-f gene deletion. (A) In situ hybridization of full-length Clr-f DIG-labelled sense and antisense probes to WT kidney tissues. DIG staining of proximal tubular cells (white arrowheads), and podocytes (black arrowhead) indicated. Scale bars represent 100 μm in upper panels and 25 μm in lower panels. (B) Schematic of genetic strategy to ablate the Clr-f gene exons 3, 4, and part of 5 with targeting vector by homologous recombination. Southern blot analysis 5′ and 3′ probe targets, and BamHI and PstI restriction enzyme digestion sites, used to confirm ES cell clone targeting, are shown. Locations of PCR primer annealing sites for genotype validations performed in Fig. S1 are shown in red and black. Locations of Clr-f RT-PCR primers (I) and (II) annealing sites in exons 1 and 5 are shown. (C) RT-PCR analysis of the indicated transcript expression in various organs. Heart tissue-derived cDNA was used as a negative control for Clr-f transcript expression. Two primer sets were used to amplify the Clr-f transcript: the binding sites for PCR primers (I) or (II) are shown in the Clr-f ^WT panel B. Cropped gel images of separate gels are shown. Full-length gel images are shown in Fig. S3A. (D) IF of the renal cortex and small intestine of WT and Clr-f^−/− mice using the 10A6 antibody. Glomeruli are indicated in kidney IF panels, Clr-f staining is shown in green, nuclei are shown in red. Scale bars represent 40 μm.