Development of Genetic Tools for a Short-lived Fish Model in Aging Research

描述（由申请人提供）：具有实验可操作基因组的动物对衰老研究非常宝贵。在蠕虫、苍蝇和小鼠等模式生物中，特异性添加或删除基因的能力使得描述特定基因在寿命调节中的作用成为可能。虽然这些动物模型已经并将继续为我们对衰老生物学的理解做出贡献，但每种现有动物模型所提供的实验优势范围仍存在差距。该项目将重点研究一种新的、有前途的脊椎动物模式生物，即一年生绿松石鳉（Nothobranchius furzeri），它的寿命只有几个月。furzeri N. furzeri提供了许多额外的优势，比如一次交配就能产生许多兄弟后代，维护成本低，易于遗传筛选，并且它们的卵可以在低温下干燥储存长达一年。尽管有这些优点，N. furzeri模型并没有在衰老研究领域得到广泛应用。这在一定程度上是因为基因操作实验不可能在这个物种中进行。最近，我的实验室已经成功地开发了在这个物种中产生稳定和可诱导的转基因的方法。根据我们对鱼类生物学和IGF信号通路的研究经验，本应用程序的目标是开发用于进行功能丧失研究的新遗传工具，并使用它们来测试胰岛素样生长因子结合蛋白-3 (IGFBP-3)，一种主要的IGFBP，通过依赖和/或不依赖IGF的机制调节寿命的假设。IGFBP-3通过紧密结合IGF并仅在特定条件下释放IGF来调节IGF的可用性。IGFBP-3也具有独立于igf的作用。这种保守的多功能蛋白可能在调节衰老中起关键作用。在目标1中，我们将开发遗传工具并使用它们来确定IGFBP-3在寿命调节中的作用。目的2将通过产生和研究表达野生型或突变型IGFBP-3的可诱导转基因系来研究IGFBP-3配体结合和核定位的功能意义。这些研究的完成将为IGFBP-3在脊椎动物寿命中的作用提供新的见解。预期的结果也将导致急需的方法和遗传工具的发展，以敲除感兴趣的基因或在这种新兴的脊椎动物模型中以组织特异性和暂时受限的方式诱导基因表达。本项目开发的方法和转基因工具将广泛传播和分发给科学界。这些遗传工具和方法，结合N. furzeri的独特特征和极短的寿命，将为研究开辟许多新的途径，并促进脊椎动物衰老生物学的新发现。更好地了解衰老调节的生物学将可能导致诊断和治疗工具的发展，从而延长美国老龄化人口的健康寿命

DESCRIPTION (provided by applicant): Animals with experimentally manipulable genomes have been invaluable for aging research. In model organisms such as worms, flies, and mice, the ability to specifically add or delete a gene enables the characterization of the roles of specific gene(s) in lifespan regulation. While these animal models have contributed, and will continue to contribute, to our understanding of aging biology, there are gaps in the range of experimental strengths provided by each of the existing animal models. This project will focus on a new and promising vertebrate model organism, the annual turquoise killifish (Nothobranchius furzeri), which has a lifespan of just several months. N. furzeri provides many additional advantages, such as the ability to produce many sibling progeny from a single mating, low cost of maintenance, amenability to forward genetic screens, and the ability of their eggs to be stored dry at low temperatures for as long as a year. Despite these advantages, the N. furzeri model has not been widely used in the field of aging research. This is in part because genetic manipulation experiments were not possible in this species. Recently, my lab has succeeded in developing methods for generating stable and inducible transgenesis in this species. Drawing on our research experience with fish biology and the IGF signaling pathway, the goal of this application is to develop new genetic tools for conducting loss-of-function studie and to use them to test the hypothesis that insulin-like growth factor binding protein-3 (IGFBP-3), a major IGFBP, regulates lifespan via IGF-dependent and/or -independent mechanisms. IGFBP-3 regulates IGF availability by binding IGF tightly and releasing it only under certain conditions. IGFBP-3 also has IGF-independent actions. It is possible that this conserved and multifunctional protein may play key roles in modulating aging. In Aim 1, we will develop genetic tools and use them to determine the role of IGFBP-3 in lifespan regulation. Aim 2 will investigate the functional significance of ligand-binding and nuclear localization of IGFBP-3 by generating and studying inducible transgenic lines that express either wild type or a mutant form of IGFBP-3. The completion of the proposed studies will provide novel insights into the roles of IGFBP-3 in the longevity of vertebrates. The anticipated results will also lead to the development of much needed methods and genetic tools to knockout a gene of interest or to induce gene expression in a tissue-specific and temporally restricted manner in this emerging vertebrate model. The methods and transgenic tools developed in this project will be broadly disseminated and distributed to the scientific community. These genetic tools and methods, combined with the unique features and the extremely short-lifespan of N. furzeri, will open many new avenues for investigations and should facilitate new discoveries in vertebrate aging biology. A better understanding of the biology of aging regulation will likely lead to the development of diagnostic and therapeutic tools that will increase the healthy lifespan of the aging population in the United States.