Gene delivery for Anopheles mosquitoes

项目摘要 人类疟疾是由以下原因引起的，它在全世界造成了极高的死亡率、发病率和经济损失： 疟原虫属的原生寄生虫，通过按蚊传播。 传统控制方法的失败刺激了人们努力开发新的策略来控制 传播疟疾的蚊子，特别是按蚊。冈比亚。虽然按蚊物种的转基因操作 已经完成了，安的常规操作。冈比亚已被证明具有挑战性， 因此在非专业实验室中不能广泛使用。开发新颖，易于使用的工具， 常规正向遗传学研究。冈比亚对控制疟疾的实用战略和基本 对这一重要蚊子媒介物种的遗传学和宿主/寄生虫相互作用进行研究。 浓核病毒，或“浓核病毒”（DNV），是细小病毒科中的单链DNA病毒 具有非常小的基因组（4-6 kb），其在5 ′和3 ′处的末端发夹结构的两侧 形接头.可以将整个病毒基因组置于感染性质粒中， 在转染到适当的细胞系中后产生。在我们的实验室里，我们只发现了已知的 浓核病毒（AgDNV）能够在冈比亚按蚊中感染和传播。AgDNV复制品 优选在成年蚊子组织中达到非常高的滴度，但完全非致病性。我们已经开发 并验证了使用AgDNV表达分泌的效应子或microRNA的新技术，这些效应子或microRNA可以调节或 改变按蚊基因表达的模式。我们的总体假设是，AgDNV可以用于过表达或 在冈比亚按蚊中敲低感兴趣的特定基因的表达，导致基本和 应用的重要性。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安氏按蚊常规正向遗传学研究。冈比亚，侧重于调制 恶性疟原虫感染/传播; 2）开发基于AgDNV的系统，用于常规逆转录病毒， 冈比亚按蚊的遗传学，重点是恶性疟原虫感染/传播和蚊子的调节 3）表征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。 这项研究将导致开发一种新的工具集，用于解决按蚊的基本问题， 疟原虫生物学，以及开发人类疟疾的潜在控制剂。

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.