A systematic RNAi-based map of C. elegans embryogenesis

描述(由申请人提供)： 人类基因组计划的下一个主要目标是确定其编码的所有基因的功能。可以说，最大的挑战在于确定大量基因的体内功能以及它们如何协调细胞的复杂过程，而动物模型可以在指导人类基因组的体内功能注释方面发挥重要作用。线虫是研究细胞生物学和动物发育的分子遗传学的重要模式系统，也是功能基因组研究的主要模式，特别是早期胚胎是研究基本细胞生物学和发育过程的优秀系统。RNA干扰(RNAi)是一种快速鉴定基因在体内功能的反向遗传学方法，到目前为止，在线虫中进行的几次大规模RNAi扫描已经识别了1500多个胚胎发育所需的基因。通过使用早期胚胎发生的时间推移显微镜系统地详细注释这些基因的功能，已经发现了200多个基因的特定细胞作用；然而，估计至少还有1000个对胚胎发育至关重要的基因仍有待识别。现在需要的下一步是提供在这个系统中发挥作用的所有基因的系统、详细的功能表征--体内功能的“表型图”，为综合系统生物学方法奠定基础。 该项目的目标是使用RNAi制作线虫早期胚胎发生的高分辨率表型图：详细描述早期胚胎中基本细胞、亚细胞和发育过程所需的所有基因的功能。为了实现这一目标，ORFeome计划克隆的所有大约12,000个有效ORF的单基因RNAi将被用于检测胚胎致死性，并使用DIC光学技术对早期胚胎发生进行系统的详细表型分析。在线数据库RNAiDB将用于这项研究的所有方面：数据收集、评分、分析和分发。将进行基于表型的生物信息学分析，包括基于表型数据的基因聚类和与其他类型的功能基因组数据的整合，以识别具有相似功能的基因组，并扩展功能注释，特别是针对未知蛋白质。这些分析的结果将被用来选择一组基因进行进一步研究，使用一组亚细胞标记来分析特定的过程(例如染色体分离、细胞骨架组织、细胞极性和细胞命运)。这些二级研究将被用来检验从早期的表型和生物信息学分析中产生的假说，以及产生关于特定基因组功能的更深入的数据。由于这些基本功能中的许多都是由高度保守的蛋白质执行的，因此在这个项目中收集的数据将立即有用于指导人类和其他基因组的功能注释。

DESCRIPTION (provided by applicant): One of the next major goals of the Human Genome Project is to identify the function of all the genes it encodes. Arguably the greatest challenge lies in determining the in vivo functions for large sets of genes and how they orchestrate the complex processes of the cell, and animal models can play a significant role in guiding in vivo functional annotation of the human genome. C. elegans is well established as an important model system in which to study the molecular genetics of cell biology and animal development and has also become a leading model for functional genomic research; the early embryo in particular is an excellent system in which to study basic cell biological and developmental processes. RNA interference (RNAi) is a rapid reverse genetics approach to identify the in vivo functions of genes, and several large-scale RNAi scans in C. elegans have so far led to the identification of over 1,500 genes required for embryogenesis. By systematically annotating the functions of such genes in detail using time-lapse microscopy of early embryogenesis, specific cellular roles for over 200 genes have been discovered; however, it is estimated that at least an additional 1000 genes essential for embryonic development remain to be identified. The next step now required is to provide a systematic, detailed functional characterization of all the genes that play a role in this system - a "phenotypic map" of in vivo functions to lay the foundation for integrative systems biology approaches. The goal of this project is to use RNAi to produce a high-resolution phenotypic map of early embryogenesis in C. elegans: a detailed functional description of all the genes required for basic cellular, subcellular, and developmental processes in the early embryo. To accomplish this goal, single-gene RNAi of all approximately 12,000 validated ORFs cloned by the ORFeome project will be performed to assay embryonic lethality and carry out systematic detailed phenotypic analysis of early embryogenesis using DIC optics. An online database, RNAiDB, will be used for all aspects of this study: data collection, scoring, analysis, and distribution. Phenotype-based bioinformatic analysis will be performed, including gene clustering based on phenotypic data and integration with other types of functional genomics data, to identify groups of genes with similar functions and to extend functional annotations, for unknown proteins in particular. The results of these analyses will be used to select groups of genes for further study using a set of subcellular markers to assay specific processes (e.g. chromosome segregation, cytoskeletal organization, cell polarity and cell fate). These secondary studies will be used both to test hypotheses generated from earlier phenotypic and bioinformatic analyses, as well as to generate more in-depth data on the functions of particular gene sets. Since many of these basic functions are carried out by highly conserved proteins, the data gathered in this project will be immediately useful to guide the functional annotation of the human and other genomes.