1998 Global Conference Proceeding
March 02 - 04, 1998
| Conference: | 1998 Global Conference |
|---|---|
| Title: | Targeting of the V2 Vasopressin Receptor Gene in Mice |
| Authors: | Yun, June; Liu, Jie; Schoneberg, Torsten; Wess, Jurgen |
| Institutions: | NIH, National Institute of Diabetes and Digestive and Kidney Diseases, University of Maryland School of Medicine, University of Leipzig, National Institutes of Health, NIDDK |
Loss-of-function mutations within the V2 vasopressin receptor gene are known to cause X-linked nephrogenic diabetes insipidus (NDI). The availability of an animal model of X-linked NDI would greatly facilitate the testing of new therapeutic strategies; however, no such model exists at this time. We have, therefore, decided to create a mouse line carrying a targeted mutation known to cause X-linked NDI in humans within the mouse V2 vasopressin receptor gene.
Initially, the mouse V2 receptor gene was cloned from a P1 genomic library derived from the mouse strain 129/SvJ. Similar to the human V2 receptor, the mouse V2 receptor consists of 371 amino acids, and it shows 88% identity to the human V2 receptor amino acid sequence. The mouse V2 receptor gene also has a genomic structure similar to that of the human V2 receptor gene. The coding sequence is interrupted by two introns; the first intron is 355 bp in length and splits codon 9; the second is 159 base pairs in length and interrupts codon 304.
To facilitate homologous recombination in mouse embryonic stem (ES) cells, a targeting vector was constructed that contained ~7.5 kb total of homologous genomic sequences derived from the mouse V2 receptor gene. A neomycin resistance gene was inserted into intron 2 to serve as a selection marker; the neo cassette was further flanked by P1 loxP sequences, thus giving us the option of removing the neo gene by the use of P1 Cre recombinase at a later point. In addition, a Glu242->Stop point mutation was introduced into the mouse V2 receptor coding sequence. This mutation, which also occurs in NDI patients (Wildin et al., Am. J. Hum. Genet., 55, 266-277, 1994), is known to result in a truncated, functionally inactive receptor protein. Several properly targeted ES cell clones were isolated and subsequently injected into C57Bl/6 blastocysts which were then implanted into pseudopregnant foster mothers. Several highly chimeric male mice were obtained. These chimeric animals are currently being mated with wild-type (CF-1) females to achieve germline transmission of the mutant V2 receptor allele.