DETECTION OF MEGAKARYOCYTE-SPECIFIC GENES & GENES ASSOCIATED WITH BONE DISORDERS

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are goal is the isolation and characterization of megakaryocyte- specific gene products in normal and disease states. To this end we plan the following steps: (1) isolation of mRNA from peripheral blood platelets or from megakaryocytes grown in culture from peripheral blood or bone marrow; (2) reverse transcription to make cDNA; (3) a subtraction procedure to enrich for gene products that are only expressed in disease states or only in megakaryocytes; (4) cloning of these specific messages; (5) sequencing and further characterization. Each of these steps involves further technical development. We have successfully isolated mRNA from platelets, and used this mRNA as a template for RT-PCR. Initial attempts to enrich by subtraction have not yet been successful. We have also grown megakaryocytes from peripheral blood mononuclear cells purified over a ficoll gradient, and are currently optimizing methods for confirming the identity of these cells and isolating them at an acceptable purity.