Reverse genetics of mosquito systemic immunity

蚊子全身免疫的反向遗传学

• 批准号: 6873920
• 负责人: ALEXANDER SIMEON RAIKHEL
• 金额: $ 40.84万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6873920
• 关键词: Aedes; Plasmodium; chickens; cytokine; defensins; genetic regulation; genetic strain; genetic techniques; genetically modified animals; high throughput technology; humoral immunity; immune response; immunodeficiency; immunogenetics; laboratory rabbit; laboratory rat; microarray technology; microorganism immunology; molecular genetics; nuclear factor kappa beta; protease inhibitor; serine proteinases; technology /technique development; transcription factor

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.

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应答的分子机制。在本申请中，将开发反向遗传学工具来研究这些机制。我们已经产生了一种转基因的、显性阴性的Relish蚊子株（RMID），其在服用血粉后变得对革兰氏阴性细菌免疫缺陷（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，以评估AMP的性质，在各种遗传杂交涉及转基因敲除免疫途径。我们将研究IMD/Relish通路在抗菌和抗寄生虫免疫中的作用。我们将测试这一假设，即Relish在蚊子的体液免疫中起着关键作用：将详细描述Relish基因敲除和血粉激活、过表达的Relish在转基因蚊子中对免疫应答的影响。我们将检验这样的假设，即Dorsal是Toll途径中的关键转录因子，在全身免疫中具有双重作用：（1）激活抗细菌和抗真菌效应器，以及（2）负责产生调节分子，例如细胞因子、丝氨酸蛋白酶原及其抑制剂（serpins）。将产生稳定的、显性阴性的和血粉活化的、过表达的背纹伊蚊菌株。IMD/Relish和Toll途径之间的相互作用将通过建立遗传稳定的Relish/Dorsal杂交菌株进行研究。高通量的基因表达分析，利用cDNA微阵列，将被用来确定由两个研究的途径，诱导感染细菌，真菌和疟原虫病原体的影响和信号转导基因的剧目。基因稳定的敲除和过表达转基因菌株与改变的主要免疫途径，结合感染，将在微阵列测定中使用，以建立病原体-途径-效应物/信号传导基因之间的调控联系。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，预计将产生有关这些途径调控的关键基因的信息。