Adult genome-wide phenotypic analysis of molecularly defined mutant genes

描述（由申请人提供）：尽管人类基因组包含大约20,000个基因，但通过功能丧失分析，仅在脊椎动物中检查了一小部分基因的功能。其他基因也在哺乳动物细胞培养系统中进行了功能检测。然而，基因功能的最终测试是动物模型敲除，其中基因功能与细胞培养中发现的基因功能惊人地不同。斑马鱼为人类的发育和疾病提供了有价值的脊椎动物模型。基于基因组突变和基于表型的基因突变鉴定的斑马鱼正向遗传筛选已成为基因发现和基因功能分析的有力方法。这种方法的力量要求突变基因被分子识别，通常是通过位置克隆，这需要大量的努力。此外，这种方法不能识别基因组中许多基因的功能，因为正向筛选是通过特定表型测定来设计的，并不是分析所有基因功能的广泛方法。评估脊椎动物所有基因功能的一种更快速、更经济的方法是对反向遗传方法产生的基因突变进行广泛的表型分析。几个实验室目前正在资助在斑马鱼中产生100万个分子定义的基因突变。这些实验室正在检查每个品系的胚胎/早期幼虫表型，并发现75%的幼虫可以存活。然而，基因功能并没有在早期幼虫中结束。重要的是，人类疾病通常在青少年和成人中表现出来。这一建议假设，显著部分的突变基因存活作为早期幼虫将显示功能，在后期幼虫到成虫阶段。为了研究它们的功能，我们将对1500个这样的突变基因进行后期幼虫和成虫表型筛选，扩大对胚胎发生过程中没有明显功能的约75%的基因功能的认识。纯合子突变的成年将进行形态学检查，通过x射线分析来调查骨骼，以及生育表型。对于导致成人表型的突变基因，将在整个动物切片上进行组织学分析，以在细胞水平上研究内部器官和组织缺陷。对于在成年之前致死但在早期幼虫阶段之后的突变，将确定致死的时间框架，并通过骨骼x射线和组织学分析对突变的幼虫或幼体进行形态学检查，以调查缺陷的性质。拟议的研究将为科学界提供1500个在早期发育过程中没有明显功能的基因的基线信息。表型数据的资源将为未来个体研究者对基因功能的深入研究提供一个起点。本研究将破译大量脊椎动物基因的成体功能，为斑马鱼体内的许多人类疾病基因提供模型。参与器官功能和发育、骨骼和骨骼形成以及生育的基因是许多青少年和成人发病的基因之一。

DESCRIPTION (provided by applicant): Although the human genome contains about 20,000 genes, the function of only a small fraction have been examined in a vertebrate animal through loss of function analysis. Additional genes have been examined functionally in mammalian cell culture systems. However, the ultimate test of gene function is an animal model knockout where gene function can be surprisingly distinct from that found in cell culture. The zebrafish has provided a valuable vertebrate model for human development and disease. Forward genetic screens in the zebrafish based on mutagenesis of the genome and phenotype-based identification of gene mutations have been a powerful method of gene discovery and gene function analysis. The power of this approach requires that mutant genes be molecularly identified, typically through positional cloning, which requires substantial effort. Furthermore, this approach will not identify functions for many genes in the genome, because forward screens are by design biased by a specific phenotypic assay and are not broad based methods of analysis of all gene functions. A far more rapid and cost efficient method of assessing all gene functions in a vertebrate is a broad-based phenotypic analysis of gene mutations generated by reverse genetic approaches. Several laboratories are currently funded to generate >10,000 molecularly-defined gene mutations in the zebrafish. These labs are examining embryonic/early larval phenotypes for each line and have found that 75% are viable as larvae. Gene function, however, does not end in early larvae. Importantly, human disease typically manifests itself in juveniles and adults. This proposal postulates that a significant fraction of the mutant genes viable as early larvae will display functions during late larval to adult stages. To investigate their functions, 1500 such mutant genes will be screened for late larval and adult phenotypes, expanding the knowledge base of gene function for the ~75% of genes with no evident functions during embryogenesis. Homozygous mutant adults will be examined morphologically, by X-ray analysis to investigate the skeleton, as well as for fertility phenotypes. For mutant genes that cause an adult phenotype, histological analysis will be performed on entire animal sections to investigate internal organ and tissue defects at the cellular level. For mutations lethal prior to adulthood but following early larval stages, the timeframe of lethality will be determined and mutant larvae or juveniles examined morphologically, by X-ray of skeleton and histological analysis to investigate the nature of the defect. The proposed studies will provide a baseline of information to the scientific community for 1500 genes with no apparent function during early development. The resource of phenotypic data would then provide a starting point for future in-depth studies of gene function by individual investigators. This study will decipher the adult functions of a large number of genes in vertebrates, which will provide models for many human disease genes in the zebrafish. Genes acting in organ function and development, bone and skeletal formation, and fertility are among the many juvenile and adult onset disease genes expected.