A Functional Screen for Proteins that Alter Cell Fates

描述(申请人提供)：完整的人类序列 和小鼠基因组即将到来，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 许多不同和独特的方法。这些功能方法目前 包括用于相互作用蛋白质双杂交筛选、用于 可视化表达复杂性，以及其他旨在阐明 专门化职能。我们已经开发了一种基于功能的分析方法， 我们将利用它来识别可能会改变的数百种分子 特定的细胞命运反应。 我们的主要关注点是理解形成图案所需的信号 在小鼠的原肠形成过程中指定不同的细胞命运。小白鼠 原肠作用，甚至比两栖动物和硬骨鱼的原肠作用更甚，是一个时期。 巨大的分化和增长。在这一阶段，小鼠胚胎 从只有两种细胞类型过渡到拥有数百种细胞类型。尽管有一个 令人难以置信的丰富的细胞信号来源，小鼠的原肠胚尚未被使用 由分子生物学家挖掘分子。这主要是由于规模的原因 (100uM)和无法接触到小鼠的原肠胚，因此排除了 生物化学、胚胎学和分子检测的有效应用。 我们已经设计了一种屏幕，它可以点击与细胞有关的分子的身份 小鼠原肠发育过程中的命运规范。这种方法提供了随机 将小鼠原肠胚库中的cDNA组合到更容易处理的 非洲爪哇胚胎。然后我们观察这些胚胎特定标志物的变化。 基因表达，表明细胞命运发生了积极或消极的变化。通过 继续对该屏幕进行“试运行”，我们已经确定了16个 其中8个分子的功能未知。在这里，我们将开始一项 高通量筛选以阐明改变神经、肌肉、 内皮细胞、血液细胞和内皮细胞的命运。在完成这笔赠款时 我们将提供估计为200个的序列、功能和表达数据 以前未探索过的分子。

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.