This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Introduction: The mouse is a critical genetic system to understand in vivo gene function and to develop animal models of human disease. New methods will be needed to further advance the utility of the mouse as a genetic system. In particular, the mouse genetics community has lacked a broadly applicable means of performing phenotypic screens to identify mutations in specific biochemical or genetic pathways. The development of cell-based phenotypic screens in mouse embryonic stem (ES) cells could provide the critical methodology for focused mutation screens. The TGF-beta-related signaling pathways would be excellent candidates for directed phenotypic screens in ES cells. These signaling pathways play a critical role in a variety of disease states, including tumor progression. A directed mutation screen of TGF-beta-related signaling processes would generate valuable animal models to further understand the regulation of these pathways. Many of the components that mediate TGF-beta signaling are present on mouse chromosome 18, suggesting the possibility of a chromosomal-directed phenotypic screen for TGF-beta signaling mutations.Methods: A screening strategy will be developed for identifying ES cells that carry chemically induced mutations in the immediate early response components of TGF-beta superfamily signaling on mouse chromosome 18. A luciferase based reporter will be used as a readout of the responsiveness of ES cells to TGF-beta signaling. Mutagenesis will be accomplished with ethylnitrosourea, a highly effective mutagen in ES cells. To resolve recessive mutations, a Cre-loxP-mediated mitotic recombination system will be utilized to generate cells that are homozygous for a mutagenized chromosome 18. Cell-based mutation screening strategies as developed in this proposal can be applied to any active biochemical pathway in mouse ES cells, to generate mouse lines for analysis in vivo.
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