The core will function as a service facility to investigators interested in using mouse models of ES cell differentiation to evaluate endothelial-specific gene expression during the process of endothelial differentiation. The in vitro differentiation of embryonic stem cells is a classic model of cellular differentiation that recapitulates many of the events that occur during normal embryonic development. A mayor focus of this Program Project is the identification of molecular mechanisms underlying endothelial specific gene expression. The differentiation of murine ES cells into embyroid bodies will serve as a model system for defining the molecular mechanism required for endothelial-specific gene expression in the developing embryo. This model has been well characterized and recapitulates many of the events that occur during normal mouse embryogenesis. Reproducible sequential expression of endothelial specific genes accompanied by the formation of endothelial cells and primitive vascular structures can be observed in this model by day 10 of differentiation. This model will allow the determination of which regulatory elements from the endothelial-specific genes, using Hprt-targeted constructs employing selected regulatory elements from the endothelial-specific genes to direct LacZ expression. In addition, the embryoid model will serve to identify novel surface markers that mark the transition from an undifferentiated ES cell toward a fully differentiated endothelial cell and facilitate theidentification of better markers of endothelial progenitor cells. Specific subsets of cells will beseparated by flow cytometry, allowing the identification of additional genes enriched in subsets of cells by microarray analysis, including the identification of potential novel transcriptional regulators of endothelial differentiation. Finally, the isolation and separation of particular subsets of cells of the endothelial lineage can be tested for their ability to promote angiogenesis in animal models of myocardial ischemia and infarction.
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