FUNCTIONAL GENOMICS OF THE BETA-CELL

糖尿病是一个严重的健康问题，在美国影响大约1600万人。 未来糖尿病的治疗方法将大大受益于对表达谱的全面了解 在正常和病理条件下的[3-细胞]和差异表达基因的功能注释。 该应用程序的目标是汇集三个实验室的互补专业知识， 令人兴奋的新资源-超过7,700个独特的cDNA克隆由先前的NIDDK资助的财团， “发育中的内分泌胰腺的功能基因组学”。我们的目标有三个方面： 建立一个大的cDNA微阵列，通过结合7,700个非- 用我们目前的PancChip 2.0的3，400个克隆来复制上述冗余cDNA。我们将使用这个微阵列 筛选6种干扰[3-细胞功能]的范例，以确定在目标2中进一步分析的候选基因 和3. Aim 2将从我们的收藏中选择1，000个cDNA克隆转移到FLEXGene库中。这 库将允许cDNA高通量转移到多个表达载体中。此外，我们将 选择用于产生抗血清的抗原以衍生13-细胞及其前体的标记抗体。在目标3中， 将对500个选定的cDNA进行功能评估，以确定它们在3-细胞生物学中的潜在作用。克隆转移到 将FLEXGene储存库和序列验证（Aim 2）亚克隆到腺病毒载体中，以实现高效表达。 INS-1细胞的转导。在某些情况下，差异表达基因的序列将用于设计表达载体。 干扰RNA（RNAi）寡核苷酸以允许抑制靶基因表达。调制的效果 然后在各种β-细胞功能模型中测试靶基因表达。候选cDNA在以下中呈阳性： 将在腺病毒转导的胰岛和/或转基因动物中进一步评价该筛选。在此过程中发现的基因 时尚可能成为候选药物靶点，或可用于开发基于细胞的胰岛素的替代细胞 替代疗法这个项目将作为一个有价值的基因发现的努力，将补充该计划 由NIDDK资助的[3-细胞生物学联盟]实施。通过该项目产生的新资源将 提供给NIDDK资助的生物技术中心和糖尿病研究社区。

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.