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蚊子埃及埃及埃及免疫反应的分子机制。在此应用程序中，将开发反向基因苯二醇工具来研究这些机制。我们已经产生了一种转基因，显性阴性的蚊子菌株（RMID），该蚊子在服用血液后会免疫缺乏革兰氏阴性细菌（Shin等，2003）。后者的成就为研究蚊子免疫的利用打开了利用可遗传的倒遗传学方法。 RMID转基因蚊子为评估涉及免疫途径的转基因敲除的各种遗传杂交中AMP的特性提供了宝贵的工具。我们将研究IMD/Relish途径在抗菌和抗寄生虫免疫中的作用。我们将检验以下假设，即在蚊子的体液免疫力中起着关键作用：柔和的敲除和血液的激活，过表达的转基因蚊子对免疫反应的效果将以详细为特征。我们将研究以下假设：背侧是TOLL途径的关键转录因子，在系统性免疫中具有双重作用：（1）激活抗细菌和抗真菌效应子，以及（2）负责监管分子的产生，例如细胞因子，丝氨酸促甲氨酸促蛋白促蛋白，丝氨酸促抑制剂和其抑制剂（Serpins）。将产生稳定，显性阴性和血液激活的，过表达的背部疾病。 IMD/Relish和Toll途径之间的相互作用将通过建立遗传稳定/背侧杂交菌株来研究。利用cDNA微阵列的高通量基因表达分析将用于确定效应子和信号基因的曲目，并由两种研究的途径调节，这些途径被细菌，真菌和疟原虫病原体感染引起。在微阵列测定中将采用遗传稳定的敲除和过表达的转基因菌株，并与感染结合使用，并将使用感染，以在病原体 - pathway-effector/信号传导基因之间建立调节性联系。与免疫反应的基因组分析相结合的遗传稳定显性阴性和过表达菌株的利用有望产生有关这些途径调节的关键基因的信息。