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

点击翻译按钮获取中文摘要

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.