A Functional Screen for Proteins that Alter Cell Fates

改变细胞命运的蛋白质的功能筛选

• 批准号: 6708047
• 负责人: Julie C Baker
• 金额: $ 45.8万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6708047
• 关键词: Xenopus; animal genetic material tag; apoptosis; biological signal transduction; blood cells; cell differentiation; cell type; cytogenetics; embryogenic cleavage; endoderm; gene expression; genetic library; genetic markers; high throughput technology; histogenesis; in situ hybridization; laboratory mouse; mesoderm; muscle cells; neurons; nucleic acid sequence; protein structure function

DESCRIPTION (provided by applicant): With the complete sequence of the human and mouse genomes on the horizon, the critical next step in genomic analysis is to provide functional criteria for the newly discovered proteins. Understanding the diverse protein functions present within complex genomes must encompass many different and unique approaches. These functional approaches currently include two-hybrid screens for interacting proteins, microarray techniques for visualizing expression complexity, and other assays designed to elucidate specialized functions. We have developed one such functional-based assay that we will utilize to identify potentially hundreds of molecules that can alter specific cell fate responses. Our main focus is to understand the signals necessary for patterning and specifying diverse cellular fates during gastrulation in the mouse. Mouse gastrulation, even more so than amphibian and teleost gastrulation, is a period of vast differentiation and growth. During this stage, the mouse embryo transitions from having only two cell types, to having hundreds. Although an incredibly rich source of cell signaling, the mouse gastrula has not been used by molecular biologists to mine for molecules. This is mainly due to the size (100uM) and inaccessibility of the mouse gastrula, which therefore precludes the effective use of biochemistry, embryology and molecular assays in general. We have devised a screen that taps the identity of molecules involved in cell fate specification during mouse gastrulation. This approach delivers random combinations of cDNAs from mouse gastrula libraries into the more tractable Xenopus embryo. We then observe these embryos for changes in specific marker gene expression, indicating changes - positive or negative - in cell fate. By proceeding with a "trial run" of this screen, we have already identified 16 molecules, 8 of which have no understood function. Here, we will embark on a high throughput screen to elucidate molecules that alter neural, muscle, endothelial, blood and endodermal cell fates. At the completion of this grant we will provide the sequence, function and expression data for an estimated 200 previously unexplored molecules.

描述（由申请人提供）：与人类的完整顺序 和小鼠基因组在地平线上，基因组分析的关键下一步是 为新发现的蛋白质提供功能标准。理解 复杂基因组中存在的多种蛋白质功能必须包含 许多不同且独特的方法。这些功能性方法当前 包括用于相互作用蛋白质的两个杂交屏幕，用于微阵列技术 可视化表达复杂性和其他旨在阐明的测定 专业功能。我们已经开发了一种基于功能的测定法 我们将利用来识别可能改变的数百个分子 特定的细胞命运反应。 我们的主要重点是了解图案和图案所需的信号 指定小鼠胃肠道期间的各种细胞命运。老鼠 胃肠作用，甚至比两栖动物和硬骨胃胃，这是一个时期 巨大的差异和增长。在此阶段，小鼠胚胎 从只有两种细胞类型到具有数百种细胞类型的过渡。虽然一个 小鼠胃尚未使用富含细胞信号的来源，尚未使用 由分子生物学家挖掘分子。这主要是由于大小 （100UM）和不可接受的小鼠胃肠道，因此排除了 通常，通常使用生物化学，胚胎学和分子测定法。 我们设计了一个屏幕，可以轻敲涉及细胞的分子的身份 小鼠胃肠道期间的命运规格。这种方法可随机 小鼠胃库中的cDNA组合到更可行的 Xenopus胚胎。然后，我们观察这些胚胎以改变特定标记 基因表达，表明细胞命运中的变化 - 阳性或阴性。经过 继续进行此屏幕的“试用”，我们已经确定了16 分子，其中8个没有理解的功能。在这里，我们将开始 高吞吐量屏幕以阐明改变神经，肌肉的分子 内皮，血液和内皮细胞命运。本赠款完成后 我们将为估计的200提供序列，功能和表达数据 以前未开发的分子。