Reverse genetics of mosquito systemic immunity

描述（由申请人提供）：本申请的长期目标是阐明调节埃及伊蚊免疫反应的分子机制。在此应用中，将开发反向遗传学工具来研究这些机制。我们已经培育出了一种转基因的、显性阴性的味蚊菌株（RMID），这种蚊子在摄取血液后会对革兰氏阴性细菌产生免疫缺陷（Shin et al., 2003）。后一项成就为利用可遗传的反向遗传学方法研究蚊子免疫打开了大门。RMID转基因蚊子提供了一种宝贵的工具，用于评估涉及免疫途径转基因敲除的各种遗传杂交中AMPs的特性。我们将探讨IMD/津津途径在抗菌和抗寄生虫免疫中的作用。我们将验证津津乐道在蚊子体液免疫中起关键作用的假设：津津乐道敲除和血粉激活、过表达的津津乐道在转基因蚊子中对免疫反应的影响将被详细描述。我们将研究Toll通路中的关键转录因子Dorsal在全身免疫中具有双重作用的假设：(1)激活抗菌和抗真菌效应；(2)负责产生调节分子，如细胞因子、丝氨酸前蛋白酶及其抑制剂（丝氨酸蛋白酶）。将产生稳定的、显性阴性的、血粉激活的、过表达的背伊蚊菌株。通过建立遗传稳定的flavor /Dorsal杂交品系，研究IMD/ flavor与Toll通路的相互作用。利用cDNA微阵列的高通量基因表达分析将用于确定受细菌、真菌和疟原虫病原体感染诱导的两种研究途径调控的效应和信号基因库。基因稳定的敲除和过表达转基因菌株改变了主要的免疫途径，并与感染相结合，将用于微阵列检测，以建立病原体-途径效应/信号基因之间的调节联系。利用遗传稳定的显性阴性和过表达菌株，结合免疫反应的基因组分析，有望获得有关这些途径调节的关键基因的信息。

DESCRIPTION (provided by applicant): The long-term goal of this application is to elucidate the molecular mechanisms regulating immune responses in the mosquito Aedes aegypti. In this application, reverse-genetics tools will be developed to study these mechanisms. We have already generated a transgenic, dominant-negative Relish mosquito strain (RMID), which after taking a bloodmeal becomes immune deficient to Gram-negative bacteria (Shin et al., 2003). The latter accomplishment has opened the door to utilization of the heritable reverse-genetics approach in studying mosquito immunity. The RMID transgenic mosquitoes provide an invaluable tool to assess the properties of AMPs in various genetic crosses involving transgenic knock-outs of immune pathways. We will investigate the role of the IMD/Relish pathway in antibacterial and anti-parasite immunity. We will test the hypothesis that Relish plays a key role in humoral immunity in mosquitoes: the effect of Relish knock-out and bloodmeal-activated, over-expressed Relish in transgenic mosquitoes on immune responses will be characterized in detail. We will examine the hypothesis that Dorsal, which is the key transcription factor in the Toll pathway, has a dual role in systemic immunity: (1) activating anti-bacterial and anti-fungal effectors and (2) being responsible for the production of regulatory molecules, such as cytokines, serine pro-proteases and their inhibitors (serpins). Stable, dominant-negative and bloodmeal-activated, over-expressed Dorsal Aedes strains will be generated. Interaction between IMD/Relish and Toll pathways will be investigated through establishing genetically stable Relish/Dorsal hybrid strains. High-throughput gene expression analyses, utilizing cDNA microarrays, will be employed to determine the repertoire of effector and signaling genes being regulated by the two studied pathways those that are induced by infection with bacterial, fungal and Plasmodium pathogens. The genetically stable knock-out and over-expression transgenic strains with altered major immune pathways, in combination with infections, will be employed in microarray assays to establish the regulatory links among pathogen-pathway-effector/signaling genes. Utilization of genetically stable dominant-negative and over-expression strains in combination with a genomic analysis of immune responses is expected to yield information about the key genes regulated by these pathways.