Adult genome-wide phenotypic analysis of molecularly defined mutant genes

分子定义的突变基因的成人全基因组表型分析

• 批准号: 8322799
• 负责人: Mary C. Mullins
• 金额: $ 63.49万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8322799
• 关键词: Adolescent; Adult; Animal Model; Animals; Biological Assay; Bone Development; Cell Culture System; Cell Culture Techniques; Chemicals; Communities; Data; Defect; Development; Disease; Embryo; Embryonic Development; Female; Fertility; Fertilization; Fishes; Funding; Future; Gene Mutation; Genes; Genetic; Genetic Screening; Genome; Genotype; Heterozygote; Histology; Homozygote; Human Development; Human Genome; Image; Individual; Laboratories; Larva; Mammalian Cell; Methods; Microscope; Modeling; Morphology; Mutagenesis; Mutate; Mutation; Nature; Onset of illness; Organ; Phenotype; Physiology; Research Personnel; Resolution; Resources; Roentgen Rays; Skeletal bone; Skeleton; Staging; Testing; Tissues; Vertebrates; Zebrafish; base; cost efficient; design; gene discovery; gene function; genome-wide; human disease; knockout gene; knowledge base; loss of function; male; mutant; positional cloning; skeletal; zebrafish genome

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.

描述（由申请人提供）：虽然人类基因组包含约 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的功能。其他基因已在哺乳动物细胞培养系统中进行了功能检查。然而，基因功能的最终测试是动物模型敲除，其中基因功能可能与细胞培养物中发现的基因功能惊人地不同。斑马鱼为人类发育和疾病提供了有价值的脊椎动物模型。基于基因组诱变和基于表型的基因突变识别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强大方法。这种方法的威力要求对突变基因进行分子鉴定，通常是通过定位克隆，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特定表型测定的影响，并且不是对所有基因功能进行广泛分析的方法。评估脊椎动物所有基因功能的一种更快速且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。目前有几个实验室获得资助，在斑马鱼中产生超过 10,000 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，发现 75% 的幼虫可存活。然而，基因功能并没有在早期幼虫中结束。重要的是，人类疾病通常表现在青少年和成人身上。该提议假设，早期幼虫时期存活的突变基因的很大一部分将在幼虫晚期到成虫阶段表现出功能。为了研究它们的功能，将针对晚期幼虫和成虫表型筛选 1500 个此类突变基因，从而扩展约 75% 在胚胎发生过程中没有明显功能的基因的基因功能知识库。纯合突变成虫将通过形态学检查、X 射线分析来研究骨骼以及生育表型。对于导致成年表型的突变基因，将对整个动物切片进行组织学分析，以在细胞水平上研究内部器官和组织缺陷。对于在成年之前但在早期幼虫阶段之后致命的突变，将确定致死的时间范围，并通过骨骼 X 射线和组织学分析对突变幼虫或幼虫进行形态学检查，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信息基线。表型数据资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大量基因的成年功能，这将为斑马鱼中许多人类疾病基因提供模型。作用于器官功能和发育、骨和骨骼形成以及生育力的基因是许多预期的青少年和成人发病基因之一。