Figure 4.

Generation of ZFYVE28 knockout mice. (A) Schematic drawing of the targeting strategy to generate full and conditional knockout mouse lines. The construct was targeted to surround the exon 4 of ZFYVE28 gene. The mice were crossed with Th-IRES deleter to generate a full knockout allele, and with FLP-deleter (FLP) line to generate a floxed mouse line. The floxed line was crossed with a podocin-cre line to inactivate ZFYVE28 specifically in podocytes. (B) In genotyping, full knockout allele generates a 211 bp PCR product, whereas wild type allele generates a 585 bp product. In floxed animals, an additional 688 bp product is detected. (C) In order to validate the deletion of the exon 4, we carried out PCR experiments using primes between exons 1 and 2, 3 and 4, as well as 5 and 6. In full KO animals, primer pair 3-4 cannot amplify a product in kidney and eye tissues, whereas in podocyte-specific KO animals, this pair can amplify a normal product in the eye but only a weak band is generated from kidney tissue. Full gels with ladder are shown in the Supplementary Figure 4.