FUNCTIONAL GENOMICS OF THE DEVELOPING ENDOCRINE PANCREAS

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The overall aim of this proposal is to identify and characterize genes involved in pancreatic endocrine development and function. This includes genes implicated in generating islets and the pancreas as well as genes defining the physiological function and dysfunction leading to the diseased states in type 1 and type 2 diabetes. We propose to construct comprehensive cDNA libraries from both humans and mice, using embryonic and adult pancreatic tissues. These libraries will be used to prepare cDNA chips (microarrays) that will serve as quantitative and standardized assays for gene activity. The availability of pancreatic chips will enable researchers to assay gene expression in numerous contexts (development, cancer, and diabetes) and thereby provide an effective method for gene analysis and discovery. The steps necessary to acquire DNA microarrays will be accomplished by four labs: D. Melton, Harvard; B. Brownstein, A. Permutt, and the Genome Sequencing Center (GSC), Washington University. The project will be divided into four general parts: 1) Construction of cDNA libraries and normalization by oligo hybridization (Melton), 2) Sequencing of representative cDNA clones and depositing in public databases (Permutt, Genome Sequencing Center, I.M.A.G.E. consortium); 3). Preparation of cDNA microarrays (Permutt, Brownstein); 4) Pilot experiments to ascertain the reliability and utility of microarrays for studies of pancreatic development and diabetes (Melton, Permutt, Brownstein) We propose to construct 4 libraries, pancreas pooled from various embryonic stages for mouse and human, and pooled pancreas and islet libraries from adult mouse and human. Approximately 50,000 clones will be arrayed from each library, and depending on complexity, up to 10,000 will be sequenced. All 50,000 cDNAs for each library will be spotted on DNA microarrays and pilot experiments conducted. All cDNA clones will be deposited with the I.M.A.G.E. Consortium, and all sequences placed in UniGene (NCBI) for immediate public access. At least one company (Genome Systems, St. Louis) has expressed the desire to incorporate the islet cDNAs in their expression microchips for the research community.