Reverse genetics of mosquito systemic immunity

描述(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。在这一应用中，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、显性阴性的美味蚊子品系(RMID)，这种蚊子在吃了一顿血后，对革兰氏阴性细菌产生了免疫缺陷(Shin等人，2003年)。后者的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个宝贵的工具来评估各种遗传杂交中AMP的性质，这些杂交涉及到免疫途径的转基因敲除。我们将研究IMD/AREISH通路在抗细菌和抗寄生虫免疫中的作用。我们将验证调味品在蚊子体液免疫中起关键作用的假设：将详细描述转基因蚊子中调味品敲除和血粉激活、过度表达的调味品对免疫反应的影响。我们将检验这一假设，即作为Toll途径中的关键转录因子，DRADAL在系统免疫中具有双重作用：(1)激活抗细菌和抗真菌效应器；(2)负责调节分子的产生，如细胞因子、丝氨酸酶原及其抑制物(Serpins)。将产生稳定的、显性阴性的、血液激活的、过度表达的背部伊蚊菌株。IMD/AREISE和Toll途径之间的相互作用将通过建立遗传稳定的ARISE/背部杂交菌株来研究。利用基因芯片的高通量基因表达分析将被用来确定由细菌、真菌和疟原虫感染诱导的两条被研究的途径所调控的效应基因和信号基因的谱系。基因稳定的基因敲除和过表达的主要免疫途径改变的转基因菌株，结合感染，将被用于基因芯片分析，以建立病原体-途径-效应/信号基因之间的调控联系。利用遗传稳定、显性阴性和过度表达的菌株，结合对免疫反应的基因组分析，有望获得有关这些途径调控的关键基因的信息。

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.