The goal of this proposal is to develop and experimentally validate a high throughput phenotype screening procedure, that when linked to a ethylnitrosourea (ENU) mutagenesis or a random gene-targeted program will: (1) detect mutations in genes previously unrecognized as affecting hypertension; and (2) detect mutations in genes previously unrecognized as affecting cardiac hypertrophy and cardiomyopathy. In designing the screen these investigators note that ENU-induced single base pair changes and random targeted genes will much more likely be loss of function mutations than gain of function mutations. Furthermore, the screen must be able to detect random mutations in heterozygotes, preferably prior to weaning and it must be effective regardless of whether the mutations affect blood pressure and cardiac growth in a positive or negative direction. They propose two novel approaches to identify important phenotypes from a mutagenesis screen: (1) use of surrogate markers; and (2) use of sensitizing mutants. They do not expect that a single surrogate will be adequate to detect mutations in all the presently known systems affecting blood pressure and cardiac growth. However, they postulate that a combination of surrogates will be able to detect a large proportion of mutations known to affect the primary phenotypes, i.e. blood pressure and cardiac growth. Accordingly they propose the following Specific Aims: (1) To identify surrogate markers that can reproducibly detect genes affecting hypertension or cardiac hypertrophy by systematically characterizing available adult heterozygous knock out and transgenic mice with either primary phenotype. Candidate surrogate markers will be evaluated by assaying multiple tissues using; (a) DNA microarrays; (b) real time RT-PCR; (c) activity of essential regulatory kinases; (d) urine cGMP and osmolality; and (e) plasma peptide hormones. (2) To validate whether surrogate markers identified in Specific Aim #1 can distinguish 3-week-old heterozygotes from wild type litter mates in a simulated ENU experiment. Heterozygous knock out and transgenic mice will be bred to wild-type B6 mice to produce F1 pups that are genetically identical except for the presence or absence of the mutation. Surrogate markers will be measured in multiple tissues from pre-weaned F1 progeny. (3) To test the novel strategy of using sensitizing mutations to enhance the resolution of the phenotypic screen. Surrogate marker(s) will be assayed in pre-weaned F1 test progeny generated by mating known heterozygous mutants with sensitizing mice that already have abnormal blood pressure or cardiac growth. By using this strategy, they expect to move the surrogate marker(s) into a range where mutations that either increase or decrease the phenotype can be detected. (4) To develop a high throughput method for detecting plasma peptide hormones and to refine in a high throughput mode, the surrogate marker assays used in specific aims #1, 2, and 3. (End of Abstract.)
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