EDGE CT: Development of a Molecular Toolkit for Integrative Organismal Research in the Microcrustacean Daphnia pulex

该项目旨在通过为在这一领域几乎没有经验的水蚤生物学家提供一个容易获得的分子工具箱、方法和工作库存来对研究界产生重大影响。将要开发的工具将使研究人员能够专注于任何基因，然后在相对较短的时间内确定完全基因敲除或基因序列/表达修改的分子、细胞、发育和/或行为后果。此外，还将开发工具，以评估在一小部分组织中或在有限的时间跨度内改变基因功能的后果。最值得注意的是，菌株的工程方法将极大地降低未来基因功能工作的复杂性，最大限度地减少注射试剂的需要，并确保关键库存的永久保存。此外，将开发的网络浏览器将作为一个免费可用的平台，通过该平台，研究人员可以查询关于整个物种基因组水平的分子变异的非常庞大的数据库，从而简化未来遗传转化和基因表达修改载体的设计。讲习班将有助于传播研究方法，并培训从生态学到分子/细胞/发育生物学的潜在用户。随着所提出的方法的发展，我们预计普氏杜氏菌将迅速成为综合理解生物生物学的主要模型，从细胞和发育功能到表型和生态后果。该项目的更广泛影响将包括对学生、博士后和本科生研究人员的培训、研讨会和向大型大型水蚤研究社区传播工具。该项目的目标是开发一套分子试剂和遗传库，将水生微甲壳动物Daphnia Pulex转变为有效研究基因功能在建立表型中的作用的主要模式。将开发有效敲除基因功能、修改或替换DNA序列以及建立组织特异性表达的方法。将开发具有着陆点的专门的水蚤品系，以有效插入新的基因序列，并允许针对种系和组织表达基因修改。这些结构将在恒定的遗传背景下永久保持。由于初步证据表明，这些方法所需的大部分单独步骤都在水蚤中进行，因此开发这一综合生物学平台的成功几率很高。实际上，通过开发这样的工具，我们(与研究界的其他成员一起)希望弥合分子/细胞遗传学和与自然生物多样性相关的表型变异之间的差距。许多独特的特性使杜氏杜鹃格外适合这类工作：非常短的世代时间；透明的身体；关于生态和生理的大量数据库；相当大的形态和行为可塑性；通过克隆繁殖繁殖基因型，并具有异交能力；以几乎无需维护的方式储存休眠卵子数十年的能力；以及对整个物种基因组范围的序列变异的广泛理解。该项目旨在通过为在这一领域几乎没有经验的水蚤生物学家提供一个容易获得的分子工具箱、方法和工作库存来对研究界产生重大影响。该奖项由EDGE计划和整合组织系统部门和新兴前沿部门的行为系统集群共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This project is designed to have substantial impact on the research community by providing a readily accessible molecular toolbox, methodologies, and working stocks for Daphnia biologists with little prior experience in this area. The tools to be developed will allow the investigator to focus on any gene, and then in relatively short order determine the molecular, cellular, developmental, and/or behavioral consequences of complete gene knockout or gene-sequence/expression modifications. In addition, tools will be developed to allow evaluation of the consequences of altering gene function in a small subset of tissues or for a limited time span. Most notably, strains will be engineered in ways that will greatly reduce the complexity of future work on gene functions, minimizing the need for injection of reagents, and ensuring permanent preservation of key stocks. In addition, a web browser to be developed will serve as a freely available platform through which investigators can query a very large data base on the genome-level molecular variation across the species, simplifying the design of future vectors for genetic transformation and gene-expression modification. Workshops will help disseminate the research methods and train potential users from ecology to molecular/cell/developmental biology. With the development of the proposed methods, we expect D. pulex to become established rapidly as a major model for the integrative understanding of organismal biology, from cellular and developmental function to phenotypic and ecological consequences. The broader impacts of the project will include the training of students, postdocs, and undergraduate researchers, workshops, and the dissemination of tools to the large Daphina research community.The goal of this project is to develop a set of molecular reagents and genetic stocks to transform the aquatic microcrustacean Daphnia pulex into a major model for the efficient study of the role of gene functions in establishing phenotypes. Methods will be developed for efficient knockouts of gene functions, modifications or replacements of DNA sequences, and establishment of tissue-specific expression. Specialized Daphnia strains will be developed with landing sites for efficient insertion of novel gene sequences and allowing germline- and tissue-specific expression of gene modifications. These constructs will be permanently maintained on constant genetic backgrounds. Because preliminary evidence exists that most of the individual steps required for these methods operate in Daphnia, the development of this platform for integrative biology has a high probability of success. In effect, by developing such tools, we (along with other members of the research community) hope to bridge the gap between molecular/cellular genetics and phenotypic variation associated with natural biodiversity. Many unique attributes make D. pulex exceptionally suited for this kind of work: a very short generation time; a transparent body; a substantial database on ecology and physiology; considerable morphological and behavioral plasticity; propagation of genotypes by clonal reproduction, combined with an ability to outcross; the ability to store resting eggs in a near maintenance-free manner for decades; and broad understanding of genome-wide sequence variation across the entire species. This project is designed to have substantial impact on the research community by providing a readily accessible molecular toolbox, methodologies, and working stocks for Daphnia biologists with little prior experience in this area. This award was co-funded by the EDGE program and the Behavioral Systems Cluster in the Division of Integrative Organismal Systems and the Division of Emerging Frontiers.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.