FUNCTIONAL GENOMICS OF THE BETA-CELL

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Diabetes mellitus is a significant health problem, affecting approximately 16 million people in the United States. Future therapeutic approaches to diabetes will benefit greatly from a complete understanding of the expression profile of the [3-cell under normal and pathological conditions and the functional annotation of differentially expressed genes. The goal of this application is to pool the complementary expertise available in three laboratories for mining of an exciting new resource--the more than 7,700 unique cDNAs cloned by the prior NIDDK-funded consortium on "Functional Genomics of the Developing Endocrine Pancreas". Our goals are three-fold: Aim 1 of this proposal will establish a large cDNA microarray enriched for genes expressed in the endocrine pancreas by combining the 7,700 non- redundant cDNAs described above with the 3,400 clones of our current PancChip 2.0. We will employ this microarray for the screen of six paradigms of perturbed [3-cell function to identify candidate genes to be analyzed further in aims 2 and 3. Aim 2 will transfer 1,000 selected cDNA clones from our collection into the FLEXGene repository. This repository will allow for high-throughput transfer of cDNAs into multiple expression vectors. In addition, we will select antigens for the production of antisera to derive marker antibodies of 13-cells and their precursors. In Aim 3 we will functionally evaluate 500 selected cDNAs for their potential role in [3-cell biology. Clones transferred into the FLEXGene repository and sequence verified (Aim 2) will be subcloned into adenovirus vectors to allow for efficient transduction of INS-1 cells. In some cases, the sequence of differentially expressed genes will be used for design of interference RNA (RNAi) oligonucleotides to allow suppression of target gene expression. The effect of modulation of target gene expression will then be tested in various models of [3-cell function. Candidate cDNAs that are positive in this screen will be further evaluated in adenovirus-transduced islets and/or transgenic animals. Genes identified in this fashion may become candidate drug targets or could be useful in development of surrogate [3-cells for cell-based insulin replacement therapy. This project will serve as a valuable gene discovery effort that will complement the program implemented by the NIDDK-funded [3-cell biology consortium. The new resources generated through this project will be made available to the NIDDK-funded biotechnology centers and the diabetes research community at large.