Figure 3.

Podocytes and the glomerulus mature normally in podocyte-specific knockout (KO) animals. (A) Strategy to generate podocyte-specific KO mice for Gprc5b. Mice carrying a targeting cassette for exon 2 of Gprc5b gene was crossed with a FLP-deleter line to generate a floxed allele, followed by crossing with podocin-cre line to remove exon 2 specifically in podocytes. (B) Genotyping of wild-type (+/+), heterozygote (+/−), and Gprc5b-cKO (−/−) mice. The wild-type band is 361 bp and bands for the floxed allele are 226 and 457 bp, respectively. (C) PCR in mouse glomeruli isolated from Gprc5b-cKO and control mice using specific primers for exons 2–3 and exons 3–4. In Gprc5b-cKO glomeruli no product is amplified when using primers for exons 2–3. (D) Western blotting for Gprc5b in mouse glomerulus shows a band around 40 kDa in control mice, whereas this protein is missing in glomeruli isolated from Gprc5b-cKO mice. Tubulin was used as a loading control. (E) No significant differences were observed in light microscopic examination of Gprc5b-cKO kidneys when compared with control kidneys. (F) In electron microscopic analysis, no morphologic abnormalities were detected in the glomerular filtration barrier in Gprc5b-cKO mice. Magnifications: ×200 in (E). Scale bar, 250 nm in (F).