Gene delivery for Anopheles mosquitoes

项目摘要 人类疟疾在世界范围内造成过度死亡、发病率和经济损失，其原因是 疟原虫属的原生动物寄生虫，主要由按蚊传播。 传统控制方法的失败促使人们努力开发新的战略来控制 疟疾的蚊子媒介，特别是蚊子。冈比亚亚目。而对按蚊物种的转基因操作 已经完成，例行公事地操纵一个。冈比亚已经被证明是具有挑战性的，而且技术可以做到 因此，在非专业实验室中并不广泛使用。开发新的、易于使用的工具 常规正向遗传学在一个。冈比亚对疟疾控制的应用战略和基本战略都是至关重要的 对这一重要蚊媒物种的遗传学和宿主/寄生虫相互作用的研究。 登革病毒是细小病毒科的单链dna病毒。 具有非常小的基因组(4-6kb)，其侧翼在5-质点和3-质点的末端发夹结构 结束了。整个病毒基因组可以被放入一个有感染性的质粒中，功能性病毒将从该质粒中 通过将其导入适当的细胞系而产生。在我们的实验室里，我们只确定了已知的 能在冈比亚按蚊中感染和传播的登革病毒(AgDNV)。AgDNV复制 优先在成年蚊子组织中产生非常高的滴度，但完全无致病性。我们已经开发出 并验证了使用AgDNV表达分泌型效应器或microRNA的新技术，这些效应器或microRNA可以调制或 改变按蚊基因表达模式。我们的总体假设是AgDNV可用于过度表达或 冈比亚按蚊中特定感兴趣基因的表达下调，导致基础和 应用重要性。这一总体假设将在以下具体目标中得到解决：1)开发一种 基于AgDNV的按蚊常规正向遗传学基因转导系统。冈比亚，专注于调制 恶性疟原虫感染/传播研究；2)建立基于AgDNV的常规逆转系统 冈比亚按蚊的遗传学，重点是对恶性疟原虫感染/传播和蚊子的调控 适合度；3)确定雄性蚊子生殖系统感染AgDNV的特征和数量；以及 确定利用自动传播将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，用于解决按蚊和 疟原虫生物学，以及人类疟疾潜在控制剂的开发。

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.