A Functional Screen for Proteins that Alter Cell Fates

描述（由申请人提供）：具有人类的完整序列 和小鼠基因组的研究即将开始，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不同蛋白质功能必须包括 许多不同的和独特的方法。这些功能性方法目前 包括用于相互作用蛋白质的双杂交筛选、用于 可视化表达复杂性，以及其他旨在阐明 专门功能。我们已经开发了一种这样的基于功能的测定， 我们将利用它来识别数百种可能改变 特定的细胞命运反应。 我们的主要重点是了解模式化所需的信号， 在小鼠原肠胚形成期间指定不同的细胞命运。鼠标 原肠胚形成，甚至比两栖动物和硬骨鱼类原肠胚形成更甚，是一个时期， 巨大的分化和生长。在这个阶段，小鼠胚胎 从只有两种细胞类型转变为数百种细胞类型。虽然 作为细胞信号的丰富来源，小鼠原肠胚还没有被用于 来挖掘分子。这主要是由于规模 （100 μ M）和小鼠原肠胚的不可及性，因此排除了 生物化学、胚胎学和一般分子测定的有效使用。 我们设计了一种屏幕，可以识别参与细胞增殖的分子。 小鼠原肠胚形成期间的命运特化。这种方法提供随机 将来自小鼠原肠胚文库的cDNA组合成更易处理的 爪蟾胚胎。然后，我们观察这些胚胎的变化， 基因表达，指示细胞命运的变化-阳性或阴性。通过 在“试运行”这个屏幕的过程中，我们已经确定了16个 分子，其中8个没有已知的功能。在这里，我们将开始一个 高通量筛选以阐明改变神经、肌肉 内皮细胞、血液细胞和内胚层细胞的命运。在这笔赠款完成后， 我们将提供大约200个基因的序列、功能和表达数据， 以前未被探索的分子。

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.