Gene delivery for Anopheles mosquitoes

按蚊的基因传递

基本信息

批准号：
9900713
负责人：
Jason L Rasgon
金额：
$ 38.12万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-05-10 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9900713
关键词：
Address Adult Anopheles Genus Anopheles gambiae Basic Science Biological Assay Biology Capsid Proteins Cell Line Culicidae Data Densovirus Development Economics Failure Family Female Gene Delivery Gene Expression Genes Genetic Genetic Materials Genome Human Immune Infection Infectious Agent Introns Laboratories Longevity Malaria Male Genital Organs Mediating Methodology MicroRNAs Morbidity - disease rate Mosquito Control Oocysts Parasites Partner in relationship Parvovirus Pathway interactions Pattern Phenotype Plasmids Plasmodium Plasmodium falciparum Population Promoter Regions Publishing Reproductive system Research Salivary Glands Single Stranded DNA Virus Sporozoites Structure System Techniques Technology Tissues Transduction Gene Transfection Transgenes Transgenic Organisms Tropism Vector-transmitted infectious disease Viral Viral Genome Virion Virus base fitness forward genetics in vivo interest knock-down male mortality novel novel strategies offspring overexpression reverse genetics success tool transgene expression transmission process vector vector competence vector mosquito

项目摘要

Project Summary Human malaria, responsible for inordinate mortality, morbidity and economic loss worldwide, is caused by protozoan parasites in the genus Plasmodium that are obligatorily transmitted by Anopheles mosquitoes. Failure of traditional control methodologies has stimulated efforts to develop novel strategies to control the mosquito vectors of malaria, particularly An. gambiae. While transgenic manipulation of Anopheles species has been accomplished, routine manipulation of An. gambiae has proven challenging, and the technology to do so is not broadly available among non-specialized laboratories. The development of novel, easy to use tools for routine forward genetics in An. gambiae is critical for both applied strategies for malaria control and basic research into the genetics and host/parasite interactions of this important mosquito vector species. Densonucleosis viruses, or “densoviruses” (DNVs), are single-stranded DNA viruses in the family Parvoviridae with very small genomes (4-6 kb) that are flanked by terminal hairpin structures at the 5-prime and 3-prime ends. The entire viral genome can be placed into an infectious plasmid from which functional virus will be produced upon transfection into an appropriate cell line. In our laboratory, we have identified only known densovirus (AgDNV) capable of infection and dissemination in Anopheles gambiae. AgDNV replicates preferentially in adult mosquito tissues to very high titer, but is completely non-pathogenic. We have developed and validated novel techniques to use AgDNV to express secreted effectors or microRNAs that can modulate or alter patterns of Anopheles gene expression. Our overall hypothesis is that AgDNV can be used overexpress or knock down expression of specific genes of interest in Anopheles gambiae, leading to phenotypes of basic and applied importance. This overall hypothesis will be addressed in the following specific aims: 1) Develop an AgDNV-based gene transduction system for routine forward genetics in An. gambiae, focusing on modulation of Plasmodium falciparum infection/transmission; 2) Develop an AgDNV-based system for routine reverse genetics in Anopheles gambiae, focusing on modulation of P. falciparum infection/transmission and mosquito fitness; 3) Characterize and quantify AgDNV infection of the male mosquito reproductive system, and determine the potential for using auto-dissemination to introduce AgDNV into mosquito cage populations. This research will result in the development of a novel toolset for addressing basic questions in Anopheles and Plasmodium biology, as well as the development of potential control agents for human malaria.

项目概要人类疟疾造成全世界过高的死亡率、发病率和经济损失，其原因是疟原虫属的原生动物寄生虫，必须由按蚊传播。传统控制方法的失败促使人们努力开发新的策略来控制疟疾的蚊子媒介，特别是冈比亚按蚊物种的转基因操作。已经完成，冈比亚按蚊的常规操作已被证明具有挑战性，并且需要实现的技术。因此，在非专业实验室中尚未广泛使用。开发新颖、易于使用的工具。冈比亚按蚊的常规正向遗传学对于疟疾控制和基础应用策略都至关重要。研究这一重要蚊媒物种的遗传学和宿主/寄生虫相互作用。浓核病毒，或称“浓核病毒”(DNV)，是细小病毒科的单链 DNA 病毒具有非常小的基因组 (4-6 kb)，两侧是 5 素数和 3 素数的末端发夹结构可以将整个病毒基因组放入感染性质粒中，从中产生功能性病毒。在我们的实验室中，我们只鉴定了已知的转染到适当的细胞系中时产生的。浓核病毒（AgDNV）能够在冈比亚按蚊中进行复制和感染。优先在成年蚊子组织中达到非常高的滴度，但我们已经开发出完全非致病性。并验证了使用 AgDNV 表达分泌效应子或 microRNA 的新技术，这些效应子或 microRNA 可以调节或我们的总体假设是 AgDNV 可以被过度表达或使用。冈比亚按蚊中感兴趣的特定基因的表达下调，导致基本和这一总体假设将在以下具体目标中得到解决： 1) 制定一个基于 AgDNV 的基因转导系统，用于冈比亚按蚊的常规正向遗传学，重点是调节。恶性疟原虫感染/传播；2) 开发基于 AgDNV 的常规逆转系统冈比亚按蚊遗传学，重点关注恶性疟原虫感染/传播和蚊子的调节 3) 描述和量化雄性蚊子生殖系统的 AgDNV 感染，以及确定使用自动传播将 AgDNV 引入蚊笼种群的潜力。这项研究将开发一套新颖的工具集，用于解决按蚊和按蚊的基本问题疟原虫生物学，以及人类疟疾潜在控制剂的开发。