Gene delivery for Anopheles mosquitoes

按蚊的基因传递

• 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
• 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

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.

项目摘要 人类疟疾负责世界范围内的死亡率，发病率和经济损失，是由 疟原虫中的原生动物寄生虫由蚊子蚊子必须传播。 传统控制方法的失败刺激了制定新的策略来控制该策略 疟疾的蚊子载体，尤其是。冈比亚。而肛门物种的转基因操纵 已经完成了，常规操作an。冈比亚已证明挑战，以及要做的技术 因此，在非专业实验室中并不广泛使用。新颖，易于使用的工具的开发 常规前向遗传学冈比亚对于疟疾控制和基本的应用策略至关重要 研究此重要的蚊子媒介规范的遗传学和宿主/寄生虫相互作用。 致密病毒或“致密病毒”（DNVS）是单链DNA病毒中的单链DNA病毒 具有非常小的基因组（4-6 kb），侧面是终端发夹结构，在5 p-prime和3-prime处 结束。可以将整个病毒基因组放入感染性质粒中 在翻译成适当的单元线时产生。在我们的实验室中，我们仅确定 十期病毒（AGDNV），能够在冈比亚按蚊中感染和传播。 AGDNV重复 优先在成年蚊子组织中，具有很高的滴度，但完全是非致病性的。我们已经发展了 并验证了使用AGDNV来表达可以调节或 改变肛门基因表达的模式。我们的总体假设是AGDNV可以过表达或 降低冈比亚按蚊中感兴趣的特定基因的表达，从而导致基本和 应用重要性。该总体假设将在以下具体目的中解决：1） 基于AGDNV的基因转移系统，用于常规远期遗传学。冈比亚，专注于调制 恶性疟原虫感染/传播； 2）开发基于AGDNV的系统以进行常规反向 gambiae的遗传学，重点是恶性疟原虫感染/传播和蚊子的调节 健康; 3）表征和量化男性蚊子复制系统的AGDNV感染，并 确定使用自动 - 隔离的潜力将AGDNV引入蚊子笼中。 这项研究将导致开发一种新颖的工具集，以解决atopheles和 疟原虫生物学以及人类疟疾潜在控制剂的发展。