2000 Global Researcher Conference Proceeding
March 10 - 12, 2000
| Conference: | 2000 Global Researcher Conference |
|---|---|
| Title: | Toward a mouse model of human non-X-linked NDI |
| Authors: | Yang, Baoxue; Tamarappoo, B.K.; Verkman, Alan S. |
| Institutions: | University of California, San Francisco, Oregon Health & Science University, University of California, S.F. |
We have shown that some AQP2 mutants that cause non-X-linked human NDI are misfolded, but functional[1,2]. For example, AQP2-T126M is retained in the endoplasmic reticulum of transfected mammalian cells. Also, we have shown that chemical chaperones such as glycerol and trimethylamine-N-oxide can correct AQP2 folding and trafficking defects in cell culture models, suggesting that chemical chaperones might be useful to correct the AQP2 defect in some forms of NDI. To test this possibility in vivo, a mouse model of NDI is needed. As a first step, the mouse AQP2 cDNA and gene was isolated and sequenced, and a mouse T126M ortholog was analyzed[3]. Based on the mouse AQP2 gene structure, a knock-in targeting vector was constructed using a 5 kb genomic DNA fragment containing exons 1-3. The "T126M" mutant was generated by site-directed mutagenesis. To retain the human consensus sequence for N-linked glycosylation, mutations H122S, N124S and A125T were also introduced in the targeting vector. A FseI restriction site was engineered for mouse genotype analysis. A Pol2neobpA selection cassette flanked by loxP sites was inserted into intron 2 for positive selection and a PGKtk cassette was inserted at the 3'-end of the AQP2 targeting sequence for negative selection. Embryonic stem (ES) cells (CB1-4) were electroporated with the linearized targeting construct. ES cell colonies generated by targeted replacement of mouse AQP2 with AQP2-T126M were selected by G418 and FIAU, and targeted colonies were identified by PCR and confirmed as heterozygous by genomic Southern blot analysis. Homozygous AQP2-T126M knock-in mice are being generated by intercross of the germ line heterozygous mice. The NDI mice should be useful in examining the physiology of NDI and the invivo testing of chemical chaperones and other novel therapeutics.
- Tamarappoo, B.K. and A.S. Verkman (1998). Defective trafficking of AQP2 water channels in Nephrogenic Diabetes Insipidus and correction by chemical chaperones. J. Clin. Invest. 101:2257-2267.
- Tamarappoo, B.K., B. Yang and A.S. Verkman (1999). Misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus. J. Biol. Chem. 274:34825-34831.
- Yang, B., T. Ma, Z. Xu and A.S. Verkman (1999). cDNA and genomic cloning of mouse aquaporin-2: functional analysis of an orthologous mutant causing nephrogenic diabetes insipidus. Genomics 57:79-83.