Large-scale Collect Of Mouse Cdna Sequences /Microarray

大规模收集小鼠 CDNA 序列/微阵列

• 批准号: 6969328
• 负责人: Minoru S Ko
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6969328
• 关键词: aging; biotechnology; complementary DNA; developmental genetics; functional /structural genomics; gene expression; genetic library; laboratory mouse; method development; microarray technology; molecular cloning; nucleic acid sequence; recombinant proteins; stem cells; tissue /cell culture

The rationale of this project is to develop a method to monitor the expression levels of a large number of genes in various experimental conditions and to understand global changes of gene expression patterns in development and aging. The systematic analysis of expression patterns of a large number of genes is rapidly becoming the method of choice for many laboratories to understand biological systems. Accordingly, the demand for high quality cDNA libraries and cDNA microarrays is increasing. The goals of this research project are to collect mouse cDNA clones and prepare cDNA microarrays containing as many mouse genes as possible. Previously we assembled and released two non-overlapping mouse gene sets: NIA Mouse 15K cDNA Clone Set, containing ~12,000 unique cDNA clones, and NIA Mouse 7.4K cDNA Clone Set, containing ~7400 unique cDNA clones. These clone sets have been freely distributed to the research community. During this period, we have continued to generate cDNA libraries and obtain cDNA sequences. We have analyzed all the ESTs that we have generated so far (a total of 249,200 high-quality EST sequences) and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, post-implantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. In our previous work, we developed a glass-slide microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 22,000 unique mouse transcripts, assembled primarily from sequences of stem cell and embryo cDNA libraries. We are currently working towards more comprehensive DNA microarrays containing 44,000 unique mouse transcripts/genes. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

该项目的基本原理是开发一种方法来监测各种实验条件下大量基因的表达水平，并了解发育和衰老过程中基因表达模式的总体变化。系统分析大量基因的表达模式正迅速成为许多实验室了解生物系统的首选方法。因此，对高质量cDNA文库和cDNA微阵列的需求正在增加。本研究的目标是收集小鼠cDNA克隆，并制备含有尽可能多的小鼠基因的cDNA微阵列。之前，我们组装并发布了两个非重叠小鼠基因集：NIA小鼠15 K cDNA克隆集，包含约12，000个独特的cDNA克隆，以及NIA小鼠7.4K cDNA克隆集，包含约7400个独特的cDNA克隆。这些克隆集已免费分发给研究界。在此期间，我们继续产生cDNA文库并获得cDNA序列。我们分析了迄今为止我们生成的所有EST（总共249，200个高质量EST序列），并将它们与公共序列进行聚类，以产生约30，000个小鼠基因的索引，其中包括977个先前未识别的基因。通过EST频率分析基因表达水平可以识别出表征植入前胚胎、胚胎干细胞和成体干细胞的基因，从而为这些细胞的功能特征提供潜在的标志物和线索。主成分分析确定了一组88个基因，其平均表达水平从卵母细胞到胚泡、干细胞、植入后胚胎，最后到新生组织逐渐降低。这可能是对细胞潜能的分子尺度的可能定义的第一步。在我们以前的工作中，我们开发了一个载玻片微阵列平台，其中含有原位合成的60-mer寡核苷酸探针，代表大约22，000个独特的小鼠转录本，主要由干细胞和胚胎cDNA文库的序列组装而成。目前，我们正在研究包含44，000个独特小鼠转录物/基因的更全面的DNA微阵列。在这项工作中恢复的序列和cDNA克隆为早期小鼠胚胎和干细胞中的基因功能提供了全面的资源。不受限制的社区访问资源可以加速广泛的研究，特别是在生殖和再生医学方面。