2004 Global Researcher Conference Proceeding
April 09 - 11, 2004
| Conference: | 2004 Global Researcher Conference |
|---|---|
| Title: | Generation of Germ Line Heterozygous Mice for Aquaporin-2 Conditional Knock-Out and Knock-In Models of NDI |
| Authors: | Yang, Baoxue; Verkman, Alan S. |
| Institutions: | University of California, San Francisco, University of California, S.F. |
Hereditary non-X-linked nephrogenic diabetes insipidus (NDI) is caused by mutations in the AQP2 water channel. An AQP2-T126M mutant knock-in mouse model was generated by targeted gene replacement using a Cre-loxP strategy (J. Biol. Chem. 276:2775-2779, 2001). However, the mutant mice failed to thrive and generally died by day 6 after birth, precluding testing of novel therapies in adult mice to reduce polyuria. Urine/serum analysis showed a urinary concentrating defect with severe serum hyperosmolality and renal insufficiency. To establish an adult mouse model of human autosomal NDI, a targeting vector for homologous recombination was constructed using a 5 kb AQP2 gene fragment containing exons 1-3. A Pol2neobpA selection cassette was inserted into intron 2 for positive selection and a PGKtk cassette was inserted at 3’-end of the AQP2 targeting sequence for negative selection. Two loxP sites were inserted just upstream from exon 2 and downstream from the Pol2neobpA cassette. Embryonic stem cells (CB1-4) were electroporated with 15 µg of the linearized targeting construct. Out of 192 ES cell colonies selected by G418 and FIAU, 5 targeted colonies were identified by PCR and confirmed as heterozygous by genomic Southern blot analysis. ES cells were injected into CD1 zygotes and transferred to pseudopregnant B6D2 females. Germ line transmission was confirmed by genomic PCR and Southern blot analysis. Germ line heterozygotes were mated with interferon inducible-cre mice to generate AQP2 conditional knock-out mice. The loxP-flanked AQP2 exon 2 is conditionally deleted by cre expression to produce AQP2 null mice. Conditional AQP2-T126M mutant mice will be produced by mating the AQP2 conditional knock-out mice with AQP2-T126M knock-in heterozygotes. These mouse models should be useful in studying the physiology of NDI and in in vivo testing of chemical chaperones and other novel therapeutics.
Yang and Verkman are attempting to generate a mouse model of human NDI for studying the physiology of NDI and seeking novel therapeutics. They have already developed a mouse line with specific mutation T126M in AQP2 gene. Unfortunately, these mutant mice had too severe a urine concentrating defect and died at very young age. To create adult mutant mice for testing the possibility that chemical chaperones can restore function to AQP2 mutants in vivo, they use a novel strategy to develop a mouse line that carries both a normal AQP2 gene and a mutant AQP2 gene. By this strategy, normal AQP2 gene can be conditionally deleted at adult age by a specific genetic mechanism. When the normal AQP2 gene is deleted, these mice will exhibit the symptoms that result from having a mutant AQP2. Yang, et al., performed a series of genetic manipulations and have got the germ line heterozygous mice bearing the genetic marks for deleting AQP2 conditionally.