Fig. 1.

Generation of mutant AQP2 (763–772 del) knockin mice. (A) Targeting strategy for generating mutant AQP2 (763–772 del) knockin mice. The diagram shows the wild-type AQP2 locus, the targeting construct, and the targeted locus before and after Cre recombination. The construct was designed to express a chimeric mutant AQP2 protein whose C terminus was exchanged with that of human mutant AQP2 (763–772 del). Two loxPs were inserted to flank the LacZ-Neo selective marker. The neocassette was deleted by mating the flox mice with CAG promoter Cre recombinase mice. (B) Structure of the chimeric mutant AQP2 protein expressed in the knockin mice. The C terminus of mouse wild-type AQP2 was exchanged with that of human mutant AQP2, which is thought to have a dominant-negative effect. Wild-type and mutant AQP2 antibodies were prepared to avoid cross-reactions. (C) Verification of homologous recombination by PCR of genomic DNA of the mice and amplification of the long arm of the construct using primer set F- and R-long arm was as shown in A. The 5.7-kb band is from the mutated allele. The primer set was designed not to amplify wild-type AQP2 gene. (D) Verification of homologous recombination by Southern blotting of BglII-digested genomic DNA derived from mouse tails. The 5.4-kb band is from the wild-type allele, and the 3.8-kb band is from the mutated allele. (E) Genotyping PCR after Cre recombination using a primer set flanking the remaining loxP site (F₁ and R₁). The 190-bp band represents the mutant allele containing the remaining loxP site, whereas the 90-bp band represents the wild-type allele. (F) Immunoblot of kidney homogenate probed with anti-wild-type AQP2 or mutant AQP2 antibody. Wild-type AQP2 (30-kDa band and higher glycosylated form) is expressed in both the wild-type and the heterozygous mutant AQP2 knockin mouse. Only the heterozygous knockin mouse exhibited expression of chimeric mutant AQP2 (34-kDa band and higher glycosylated form) in the kidney. H, heterozygous. (G) Immunoblot of deglycosylated proteins from papillae of heterozygous knockin mice probed with anti-AQP2 antibody, which recognizes the N-terminal portion of AQP2 (Santa Cruz Biotechnology). The relative amount of mutant AQP2 to wild-type AQP2 was estimated to be ≈30%.