This PGA proposes to continue the success of the PhysGen program by capitalizing on a variety of technological advances, extending the data by manipulating specific genes and assessing the results physiologically in three different genetic backgrounds. The """"""""functionality"""""""" of relevant genes will be tested by generating knockouts (KO) in rats, using a novel strategy that combines ENU mutagenesis with a yeast truncation assay. This technology will enable identification of animals with KOs of targeted genes of interest before weaning. The genes selected for KO have been derived from those that have been linked to heart, lung and blood disorders using molecular genetic, genomic and expression technologies during the first four years of all 11 PGA programs. In our preliminary studies, one of these genes (Avpla) was targeted and knocked out. As genomic background is shown to play a major role in the expression of phenotypes in KO mice, this PGA will attempt to make the KOs in 3 genetic backgrounds in the rat, the SS, BN, and FHH strains. The selection of these 3 strains will also enable PhysGen to leverage the enormous amount of phenotypic data generated on these strains during the first four years, notably 8,610 physiological data points per strain, including baseline and stressors, providing an unprecedented level of baseline data for a mutagenesis screen. Many traits have been demonstrated to be phenotypic extremes in two off these three strains. For example, hypertension is in the FHH and SS, but not the BN; whereas pulmonary traits! are abnormal in the BN and FHH, but normal in the SS. Knocking genes out in a disease background, as well as on a """"""""normal"""""""" model is a departure from the traditional strategy for making new disease models. Presumably removing a key gene in a disease pathway will ameliorate the trait in some cases, and exacerbate the phenotype in other cases. Testing in animals with a disease amounts to a sensitized screen, where we are looking to """"""""prevent"""""""" expression of the disease phenotype, as has been done in lower organisms. Once KOs are made on the inbred strains, multiple KO models can be rapidly made, as well as crossing the KOs into the 44 consomic strains to assess the impact of both a gene KO and a different physiological background. There are five specific aims to achieve the overall goals of this program: 1) Attempt to KO 70 genes that have been studied by the PGA that can be used in conjunction with our consomic strains, 2) characterize the KO rats physiologically, 3) participate in trans-PGA activities, 4) develop new data analysis tools for managing disparate data types, and for linking this information to the mouse and human data generated, and 5) provide an education component.
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