Figure 1

Rescue of cpk mouse phenotype by kidney-specific expression of Cys1-GFP. (A) Schematic diagram showing the Cys1-GFP transgene knock-in at the Rosa26 locus (Rosa26-Cys1-GFP allele) before (T^OFF) and after (T^ON) deletion of a PGK Neo cassette (yellow rectangle) flanked by LoxP sites (gray triangles). In the T^OFF configuration, expression of Cys1-GFP is prevented by the PGK Neo cassette. In cells expressing a Ksp-Cre transgene, Cre-mediated recombination deletes PGK Neo and Cys1-GFP is expressed (T^ON). SA: splice acceptor, PGK-Neo: Phosphoglycerate kinase promoter driving a neomycin resistance gene followed by 3 polyA signals (3XpA, red rectangle). The purple boxes flanking Cys1-GFP are attB sites. (B) PCR-based genotyping of Rosa26-Cys1-GFP, Ksp-Cre and Cys1 alleles in mice of the indicated genotypes. (C) Cys1-GFP rescue of gross phenotypes in cpk mice. Six-week-old wild-type (WT) and Cys1^cpk/cpk; Rosa26-Cys1-GFP; Ksp-Cre/ + rescued (R) mice are of equivalent size. Examination of kidneys from WT, R and cpk mice at 14 and 21 days of age show equivalent sizes of R and WT kidneys, with both markedly smaller than cpk kidneys. (D) Western blot analysis of total kidney protein from 6-week-old mice of the indicated genotypes. Mouse cystin is 145 amino acids long but migrates aberrantly at ~ 25 kDa on SDS-PAGE. Cystin-GFP (arrow, ~ 50 kDa) and endogenous cystin (arrowhead, ~ 25 kDa) were detected using polyclonal rabbit anti-cystin antibody, as previously described¹⁹. GAPDH served as an internal protein loading and transfer control. The asterisk indicates non-specific bands.