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：
8416363
负责人：
LAURENCE J ZWIEBEL
金额：
$ 20.03万
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 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8416363
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.

描述（由申请人提供）：遗传操纵昆虫疾病媒介（例如疟疾媒介蚊子蚊）冈比亚相对于目前在果蝇等模型昆虫中可用的分子工具的能力仍然是基本的。最近，人工核酸酶允许在以前缺乏有效工具的物种（例如斑马鱼和大鼠）中进行靶向基因组编辑。这些嵌合核酸酶将可编程序列特异性的DNA结合结构域与非特异性核酸酶结构域结合在一起，以在所需的基因组基因座处产生双链断裂，当不准确修复时可能会导致基因失活（“敲除”）。如果这些病变是在胚胎种系中产生的，则可能会传播目标突变等位基因。为了使Anopheles Gambiae启用这种方法，我们将设计并优化一系列定制核酸膜，以一对特征良好的气味受体（AGOR）基因在嗅觉信号转导中起着重要作用。在这些研究中，我们将比较两个不同的可编程核酸酶平台，以促进肛门种系中促进基因失活的效率，以便为这种生物体建立强大的反向遗传方法。通过评估各种Agor敲除对成人和幼虫期蚊子中化学感应反应的影响，将证明该策略的实用性。除了促进我们对这个重要疾病载体化学感应信号转导的基本知识外，拟议的研究（如果成功）应为这些和相关的基因修饰工具的实验室应用提供基础，并在各种相关的载体物种中提供基础。