The long-term objective of this renewal proposal is to understand the molecular basis of inherited platelet storage pool deficiency (SPD). A second objective is to identify and partially characterize new animal models of inherited human hemorrhagic disease. So far 14 mouse genes that cause SPD have been identified, and it is expected that human disease will also show variability. The current proposal is aimed at molecular identification, by positional/candidate gene approaches, of ruby-eye (ru) and pearl (Pe) genes. Ruby-eye is a possible mouse homolog of a recently mapped human SPD gene in Hermansky-Pudlak syndrome. Pearl is a model for some forms of night blindness.
The aims are to clone these genes. High resolution genetic maps which were constructed in this laboratory will be used to physically map and clone genomic regions immediately surrounding the SPD gene in YACs and other large cloning vectors. Mutant genes will be identified by complementation of mutant phenotypes in immortalized mutant melanocytes. Alternative gene identification approaches will utilize classical positional,/candidate cloning approaches including direct selection and/or exon trapping of expressed genes in inserts of large cloning vectors and differential display. Mutation identification will utilize single-strand conformational polymorphism and direct sequencing of expressed genes. Definitive functional identification of mutant genes will be performed by transfection of immortalized mutant melanocytes or by transgenic technology. In addition, mutants with bleeding defects will be identified by a battery of tests for common plasma factors and platelets. No gene identifications have been proposed for this portion of the proposal.
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