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Specific Aim 1: To use gene-trap techniques in embryonic stem (ES) cells to inactivate 2,500 random genes per year. We are inactivating genes in ES cells with gene-trap vectors. Our group has developed and optimized custom gene-trap vectors that are extremely efficient in inactivating genes (i.e., we have not observed "leaky" phenotypes). A browsable list of our gene-trapped ES cells is available on this website, and ES cells can be shipped to any interested investigator for the purpose of producing knockout mice.
Specific Aim 2: To use bioinformatics and gene-expression studies to gain insights into the possible importance of new genes for cardiopulmonary development and cardiopulmonary diseases.
a. To use computational approaches to identify potential functions for genes that we inactivate in ES cells. Many inactivated genes have no known function or relevance to cardiopulmonary disease. Existing databases are being screened to identify genes that appear to be expressed selectively in the heart, lung, or liver. More importantly, we are applying custom approaches in computational biology to identify likely functions for each gene that we inactivate.
b. To use in situ hybridization to assess expression patterns for many of the genes that we inactivate in ES cells.
c. To produce DNA microarrays containing these inactivated and other genes and to analyze changes in the expression of these genes during development and in the context of specific cardiopulmonary diseases. We are determining which of the "trapped" genes are up- or downregulated in the context of model cardiopulmonary diseases (e.g., asthma, pulmonary fibrosis). In addition, all the trapped genes are incorporated into a gene profiling analysis program, GenMAPP (Gene Micro-Array Pathway Profiler). This program automatically maps gene-microarray data onto biochemical pathways.
Specific Aim 3: To produce knockout mice of interest to the cardiopulmonary research community from a subset of gene-trapped ES cell clones.
a. To select several gene-trapped ES cell clones per year and produce knockout mice, for the purpose of understanding the roles of specific genes in lipid metabolism and atherogenesis.
b. To select several gene-trapped ES cell clones per year and produce knockout mice for the purpose of understanding their relevance to the pathogenesis of asthma, pulmonary fibrosis, and other common pulmonary diseases.
c. To select several knockout mice ES cell clones per year for the purpose of understanding the importance of specific genes in cardiopulmonary development.