FUNCTIONAL GENOMICS OF THE DEVELOPING ENDOCRINE PANCREAS

糖尿病是一个重大的健康问题，在美国大约有1600万人受到影响。胰岛素分泌不足是I型糖尿病的特征，而II型糖尿病则存在外周胰岛素抵抗。对胰岛发育和分化的全面了解将对体内和体外再生胰岛细胞的未来治疗方法大有裨益。我们建议建立一个功能基因组学资源，提供关于支配这一过程的复杂基因表达模式的详细信息。这项建议的具体目标1是建立标准化和消减的cDNA文库，丰富在发育中的内分泌胰腺中表达的罕见转录本，并确定以前未知的转录本的全长序列。目标2是生成代表数千个胰腺特异性或胰腺富集型转录本的cDNA微阵列。这个微阵列设施将在宾夕法尼亚大学糖尿病中心的背景下建立。这些微阵列的克隆将从三个来源获得：a)现有的商业上可用的用胰腺特异性探针筛选的微阵列，b)在Aim 1中获得的归一化新生胰岛cDNA文库，以及c)代表稀有胰腺转录本的消减cDNA文库。后者将利用单细胞的RNA扩增；这是宾夕法尼亚大学首创的，以生成特定于个别内分泌细胞类型的cdna文库。因此，我们将能够产生一个由数千个cDNA组成的复杂微阵列，这些cDNA既代表丰富的也代表稀有的胰腺mRNA。具体目标3是确定数千个基因在胰腺发育期间和携带影响胰腺发育和功能的突变的小鼠中的表达谱。我们将利用AIM 2中产生的微阵列来确定内分泌胰腺发育过程中的表达谱，包括整个胰腺和单个内分泌细胞谱系。我们将利用宾夕法尼亚大学生物信息学中心的计算机资源对产生的大量数据进行高效分析。该生物信息学数据库以及微阵列将向一般研究界提供。建立这些最先进的技术，并将它们应用于内分泌胰腺，将使主要研究人员能够制定和测试解决糖尿病病因和进展的新假说。

Diabetes mellitus is a significant health problem, affecting approximately 16 million people in the United States. Loss of sufficient insulin production by the pancreatic beta cell is the hallmark of type I diabetes, while in type II diabetes there is in addition peripheral insulin resistance. Future therapeutic approaches of regeneration of beta cells both in vivo and in vitro will benefit greatly from a complete understanding of the development and differentiation of the pancreatic islets. We propose to establish a functional genomics resource that will provide detailed information about the complex patterns of gene expression that govern this process. Specific aim 1 of this proposal is to develop normalized and subtracted cDNA libraries enriched for rare transcripts expressed in the developing endocrine pancreas and to determine full length sequences of previously unknown transcripts. Aim 2 is to generate cDNA microarrays representing thousands of pancreas- specific or pancreas-enriched transcripts. This microarray facility will be established within the context of the University of Pennsylvania Diabetes Center. Clones for these microarrays will be obtained from three sources: a) existing, commercially available microarrays screened with pancreas specific probes, b) the normalized newborn islet cDNA library obtained in Aim 1, and c) subtracted cDNA libraries representing rare pancreatic transcripts. The latter will take advantage of RNA amplification from single cells; which was pioneered at Penn, to generate cDNA libraries specific for the individual endocrine cell types. Thus we will be able to generate a complex microarray of thousands of cDNAs which represent both abundant and rare pancreatic mRNAs. Specific aim 3 is to determine the expression profile of thousands of genes during pancreatic development and in mice carrying mutations that affect pancreatic development and function. We will utilize the microarrays generated in aim 2 to determine expression profiles during the development of the endocrine pancreas, both in the entire pancreas as well as in individual endocrine cell lineages. We will utilize the computer resources of the University of Pennsylvania Bioinformatics Center for the efficient analysis of the large body of data generated. This bioinformatics database, as well as the microarrays, will be made available to the general research community. Establishing these state-of-the-art technologies, and applying them to the endocrine pancreas, will enable principal investigators to formulate and test novel hypotheses that address the causes and progression of diabetes.