Projects 1 and 2 will identify cDNAs encoding transcripts that are disrupted by chromosomal rearrangements associated with specific human birth defects. These experiments will result in the discovery of novel genes and provide circumstantial evidence for the involvement of these genes in the associated birth defects. However, because rearrangements may affect other nearby genes, these experiments will not prove causation. In addition the developmental analysis of human genes is difficult, and the lack of experimentally accessible mutants precludes ordering genes into developmental pathways. Therefore, the strategy that informs Project 3 is to use two model organisms, mouse and Drosophila, to determine whether the genes identified are likely to cause the associated birth defects and to provide insight into their developmental functions.
Three specific aims are proposed: (1) to clone and analyze the developmental expression of mouse and fly orthologs of human candidate genes in both wild type and appropriate mutant backgrounds, (2) to determine the map locations of the mouse and fly orthologs and to evaluate whether candidate mutants already exist, and (3) in highly selected cases, when there is not a pre-existing mutation and when the same human developmental phenotype is generated by more than one independent breakpoint, to prepare appropriate mouse and fly models by gene inactivation. Special consideration will be placed on the generation of mutant models for human disorders that affect the development of organs or tissues which are of special interest or for which there is pre-existing expertise in the laboratories of DGAP investigators. To obtain appropriate mouse cDNAs, we will screen embryonic libraries with human cDNAs and search existing EST databases. Both ESTs and the newly available genomic sequence will be used to obtain Drosphila orthologs as either cDNA clones or PCR products of conserved exons, as appropriate. Next, we will perform in situ hybridizations in mouse and fly to determine the developmental expression of the isolated genes. Mouse radiation hybrid panels and the complete Drosphila genomic sequence will be used to map these homologs and to evaluate whether there are existing mutants at the identified loci. Lastly, as appropriate, we will prepare loss-of-function mutations in mouse and fly and analyze the mutant phenotypes. Collectively, these studies have the capacity to delineate the developmental functions of new genes and to provide definitive proof that mutations in specific human genes produce associated birth defects. As such, they represent the functional endpoint to a well integrated, functional genomics program project.
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