This project has two major goals within the SCOR. The first is to determine the relevance of human genes identified in Project 1 and isolated in Project 2 to conotruncal morphogenesis. This will be accomplished by first determining the program and site of expression of candidate genes or genetic material in the developing mouse embryo by RT- PCR amplification, in situ hybridization and immunochemistry. The functional significance of any candidate gene to conotruncal development will be determined by blocking gene expression using anti-sense deoxyribonucleotides administered externally, injected locally or generated intracellular using viral vectors in cultured mouse embryos and by inappropriate gene expression following administration of viral vectors carrying full-length cDNA's. Disruption of conotruncal or vascular formation will be monitored anatomically and immunohistochemically using monoclonal antibodies specific to endocardium and endothelium. The second major goal is to independently identify and characterize, as described above, gene expressed during conotruncal morphogenesis by cardiac and cranial neural crest as well as within pharyngeal arches and the outflow tract of the heart in the control and mutant mouse embryos. This will be accomplished by differential comparison of RT-PCR amplified mRNA products on sequencing gels followed by PCR amplification, RACE extension and cDNA library screening of mouse libraries to produce probes for functional analysis. The behavior of cranial and cardiac neural crest from mutant mouse embryos will be evaluated by transplanting cells into mutant or control cultured mouse embryos as well as chick embryos. Upon completion of this work we will have determined the functional significance of any candidate human genes to conotruncal morphogenesis as well as independently identified new genes expressed during early heart development in the mouse that can be used as probes for cytogenetic and molecular studies in human material thereby beginning to unravel the program of gene expression and function being systematically played out for the correct formation and assembly of the conotruncal region of the heart.
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