Adult genome-wide phenotypic analysis of molecularly defined mutant genes

描述(申请人提供)：虽然人类基因组包含大约20,000个基因，但通过功能丧失分析，只有一小部分在脊椎动物身上进行了功能检测。更多的基因已经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基因敲除，其中基因功能可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一个有价值的脊椎动物模型。斑马鱼的正向遗传筛选基于基因组的突变和基于表型的基因突变鉴定，已成为基因发现和基因功能分析的有力方法。这种方法的威力要求对突变基因进行分子鉴定，通常是通过位置克隆，这需要大量的努力。此外，这种方法不会识别基因组中许多基因的功能，因为正向筛选在设计上受到特定表型分析的偏见，并不是所有基因功能的广泛分析方法。评估脊椎动物所有基因功能的一种更快速、更具成本效益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。几个实验室目前被资助在斑马鱼中产生10,000个分子定义的基因突变。这些实验室正在检查每一品系的胚胎/早期幼虫表型，并发现75%的品系可以作为幼虫存活。然而，基因的功能并不局限于早期幼虫。重要的是，人类疾病通常表现在青少年和成年人身上。这一建议假定，作为早期幼虫存活的突变基因的很大一部分将在幼虫晚期到成虫阶段发挥功能。为了研究它们的功能，将从1500个这样的突变基因中筛选出晚期幼虫和成虫的表型，以扩大~75%在胚胎发育过程中没有明显功能的基因的基因功能知识库。纯合子突变的成体将进行形态检查，通过X射线分析来调查骨骼，以及生育表型。对于导致成体表型的突变基因，将对整个动物切片进行组织学分析，以在细胞水平上调查内部器官和组织缺陷。对于在成年期之前但在早期幼虫阶段之后致死的突变，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。表型数据的来源将为个体研究人员未来深入研究基因功能提供一个起点。这项研究将破译脊椎动物中大量基因的成体功能，这将为斑马鱼中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预期的青少年和成人发病基因中的一部分。 公共卫生相关性：人类疾病通常表现在青少年和成年人身上。这项研究将破译脊椎动物中大量基因的成体功能，这将为斑马鱼中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预期的青少年和成人发病基因中的一部分。

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. PUBLIC HEALTH RELEVANCE: Human disease typically manifests itself in juveniles and adults. 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.