Generation and phenotype of mice harboring a nonsense mutation in the V2 vasopressin receptor gene
June Yun, … , Chu-xia Deng, Jürgen Wess
June Yun, … , Chu-xia Deng, Jürgen Wess
Published December 1, 2000
Citation Information: J Clin Invest. 2000;106(11):1361-1371. https://doi.org/10.1172/JCI9154.
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Article

Generation and phenotype of mice harboring a nonsense mutation in the V2 vasopressin receptor gene

Abstract

The V2 vasopressin receptor (V2R) plays a key role in the maintenance of a normal body water balance. To generate an in vivo model that allows the physiological and molecular analysis of the role of V2Rs in kidney function, we have created mouse lines that lack functional V2Rs by using targeted mutagenesis in mouse embryonic stem cells. Specifically, we introduced a nonsense mutation known to cause X-linked nephrogenic diabetes insipidus (XNDI) in humans (Glu242stop) into the mouse genome. V2R-deficient hemizygous male pups showed a decrease in basal urine osmolalities and were unable to concentrate their urine. These pups also exhibited an enlargement of renal pelvic space, failed to thrive, and died within the first week after birth due to hypernatremic dehydration. Interestingly, female mice heterozygous for the V2R mutation showed normal growth but displayed an XNDI-like phenotype, characterized by reduced urine concentrating ability of the kidney, polyuria, and polydipsia. Western blot analysis and immunoelectron microscopic studies showed that the loss of functional V2Rs had no significant effect on the basal expression levels of aquaporin-2 and the bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1). The V2R mutant mice described here should serve as highly useful tools for the development of novel therapeutic strategies for the treatment of XNDI.

Authors

June Yun, Torsten Schöneberg, Jie Liu, Angela Schulz, Carolyn A. Ecelbarger, Dominique Promeneur, Soren Nielsen, Hui Sheng, Alexander Grinberg, Chu-xia Deng, Jürgen Wess

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PCR analysis of genomic DNA prepared from tissues of V2R mutant mice to ...
PCR analysis of genomic DNA prepared from tissues of V2R mutant mice to verify Cre-mediated excision of the neo gene. Homozygous male EIIa-cre mice were bred with female V2R+/– mutant mice. Resulting V2R+/– female offspring were then interbred with wild-type male littermates to achieve germline transmission of the neo deletion. To verify the ablation of the neo gene, genomic DNA was prepared from tail (Ta), kidney (Ki), and liver (Li) samples derived from the resulting pups and subjected to PCR genotyping. The two PCR primers used for this analysis were chosen to flank the loxP-enclosed neo cassette and were located at the exon 2/intron 2 (5′-gaagctcctctggaaagtggt-3′) and intron 2/exon 3 (5′-tcctatgaagaagagagaccag-3′) junctions (see Figure 1a), respectively. After Cre-mediated excision of the neo gene (note that one 34-bp loxP sequence remains in the genome after the excision of the neo cassette), the two primers amplify a 210-bp PCR fragment, whereas the corresponding wild-type fragment is 34 bp shorter (176 bp). The absence of neo sequences in all samples was confirmed by the use of internal neo primers (data not shown). The DNAs analyzed here were taken from three different mouse pups of the indicated V2R genotypes.