Targeted gene inactivation in Anopeles gambiae via artificial nucleases

通过人工核酸酶对冈比亚按蚊进行靶向基因失活

基本信息

批准号：
8227919
负责人：
LAURENCE J ZWIEBEL
金额：
$ 25.44万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-02-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8227919
关键词：
Abbreviations Adult Affect Alleles Animals Anopheles Genus Anopheles gambiae Back Behavioral Binding Biological Assay Bite Blastoderm C2H2 Zinc Finger Cell Culture Techniques Cell Line Cells Chemicals Chimera organism Code Culicidae Custom DNA Binding Domain Development Disease Vectors Drosophila genus Elements Embryo Evaluation Foundations Frequencies Gene Family Gene Proteins Gene Silencing Genes Genetic Genome Genome engineering Genomics Goals Human Injection of therapeutic agent Insect Vectors Insecta Knock-in Mouse Knock-out Knowledge Laboratories Larva Lead Lesion Malaria Mediating Messenger RNA Methods Microinjections Modeling Modification Molecular Morbidity - disease rate Mosquito-borne infectious disease Neurons Odorant Receptors Organism Play Polymerase Chain Reaction Population RNA-Directed DNA Polymerase Rattus Receptor Gene Relative (related person)Research Reverse Transcriptase Polymerase Chain Reaction Role Series Signal Transduction Smell Perception Somatic Cell Specificity Staging System Technology Testing Transcription Coactivator Validation Yeasts Zebrafish Zinc Fingers base capitate bone design design and construction expectation in vivo insect disease interest member mortality mutant novel strategies nuclease positional cloning repaired response tool transmission process vector vector control vector mosquito

项目摘要

DESCRIPTION (provided by applicant): The ability to genetically manipulate insect disease vectors such as the malaria vector mosquito Anopheles gambiae remains rudimentary relative to the abundance of molecular tools that are currently available in model insects such as Drosophila. Recently, artificial nucleases have allowed targeted genome editing in species, such as the zebrafish and rat, that previously lacked effective tools. These chimeric nucleases combine a programmable sequence-specific DNA-binding domain with a non-specific nuclease domain to generate a double strand break at a desired genomic locus, which when imprecisely repaired can result in gene inactivation ("knockouts"). If these lesions are generated within the embryonic germline the propagation of targeted mutant alleles is possible. In order to enable this approach for Anopheles gambiae, we will design and optimize a series of custom nucleases targeting a pair of well-characterized odorant receptor (AgOr) genes that play essential roles in olfactory signal transduction. In these studies, we will compare two different programmable nuclease platforms for their efficiency in promoting gene inactivation in the Anopheles germline, with the goal of establishing a robust reverse genetic approach for this organism. The utility of this strategy will be demonstrated by evaluating the effect of various AgOr knockouts on chemosensory responses in adult and larval stage mosquitoes. In addition to advancing our basic knowledge of chemosensory signal transduction in this important disease vector, the proposed studies, if successful, should provide a basis for the laboratory- based application of these and related gene modification tools in Anopheles and a wide range of related vector species. PUBLIC HEALTH RELEVANCE: The transmission of human malaria through mosquito bites is a leading cause of worldwide mortality and morbidity despite decades of research. This project is focused on the developing genetic tools that will significantly enable mosquito research and provide a more detailed understanding of the mosquito's sense of smell. These advances could eventually lead to the development of new chemicals and approaches that reduce the transmission of malaria and other mosquito borne diseases.

描述（由申请人提供）：相对于目前在模型昆虫（例如果蝇）中可用的大量分子工具，对昆虫疾病载体（例如疟疾载体冈比亚按蚊）进行基因操纵的能力仍然处于初级阶段。最近，人工核酸酶允许对以前缺乏有效工具的物种（例如斑马鱼和大鼠）进行有针对性的基因组编辑。这些嵌合核酸酶将可编程序列特异性 DNA 结合结构域与非特异性核酸酶结构域相结合，在所需的基因组位点处产生双链断裂，如果修复不精确，可能会导致基因失活（“敲除”）。如果这些损伤是在胚胎种系内产生的，则目标突变等位基因的传播是可能的。为了使这种方法能够应用于冈比亚按蚊，我们将设计和优化一系列针对一对在嗅觉信号转导中发挥重要作用的特征明确的气味受体 (AgOr) 基因的定制核酸酶。在这些研究中，我们将比较两种不同的可编程核酸酶平台在促进按蚊种系基因失活方面的效率，目的是为该生物体建立强大的反向遗传方法。该策略的实用性将通过评估各种 AgOr 敲除对成年和幼虫期蚊子化学感应反应的影响来证明。除了提高我们对这一重要疾病载体化学感应信号转导的基础知识外，拟议的研究如果成功，还应为这些及相关基因修饰工具在按蚊和广泛的相关载体中的实验室应用提供基础物种。公共卫生相关性：尽管经过数十年的研究，通过蚊虫叮咬传播人类疟疾仍然是全世界死亡和发病的主要原因。该项目的重点是开发遗传工具，这将极大地促进蚊子研究并提供对蚊子嗅觉的更详细的了解。这些进步最终可能导致新化学品和方法的开发，以减少疟疾和其他蚊媒疾病的传播。