The central objective of the current application will be to couple molecular and genetic-based approaches to the RXRa mouse model to determine whether the relative gene dosage level of RXRa can be an important determinant of the onset of specific cardiovascular malformations, to determine whether differences in genetic background can suppress the onset of specific cardiovascular malformations, to determine if breeding into other defined transgenic or gene-targeted backgrounds can promote or suppress the onset of these malformations, to identify the target genes that are downstream from RXRa and which are affected in these specific cardiovascular malformations, and to establish a transgenic rescue system capable of evaluation the role of specific downstream target genes in the onset of specific cardiovascular malformations using mouse genetic approaches. Accordingly, the Specific Aims are: 1) To determine if there is a gene dosage effect for the onset of specific cardiovascular malformations in the RXRa deficient mice; 2) To determine if genetic background and other modifying genes can promote or suppress the onset of specific cardiovascular malformations in the RXRa mice; 3) To identify a subset of the downstream target cardiac genes that are aberrantly expressed (mis, non, or over-expression) in the RXRa deficient mice and which closely correlate with the onset of specific cardiovascular malformation; and 4) To establish a transgenic approach for rescue of the cardiovascular defects in RXRa gene-targeted mice designed to ultimately allow an examination of the rescue capabilities of the downstream target genes, identified in Specific Aim III. Through the systematic analysis of the role for a specific member of the retinoid receptor gene family (RXRa), these studies should provide a molecular framework for the understanding how vitamin A deficiency can produce specific congenital heart malformations. By crossing into other genetic backgrounds and other transgenic/gene-targeted mouse lines, these studies should lead to the generation of strains of mice that spontaneously display defined cardiovascular malformations which should be valuable to explore the interaction of vitamin A/retinoid-dependent pathways with other nutritional deficiencies. In the event that downstream target genes are identified which are influenced by specific nutrients, (e.g., extracellular matrix genes that are vitamin C dependent, transcriptional factors that are sensitive to relative levels of zinc, etc.), these would provide insights as to which potential nutritional deficiencies might augment the onset of congenital heart malformations I the in vivo context. Finally, these studies should also lead to a system that employs genetic rescue to exact cause-effect relationships between a specific downstream target gene and specific subsets of cardiovascular malformations.
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