MOSQUITO TRANSFORMATION WITH NOVEL RETROVIRAL VECTORS

新型逆转录病毒载体对蚊子的转化

• 批准号: 2074510
• 负责人: Jane L. Burns
• 金额: $ 25.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2074510
• 关键词: Aedes; Flaviviridae; Retroviridae; disease vectors; gene expression; genetic manipulation; genetically modified animals; polymerase chain reaction; tissue /cell culture; transfection /expression vector; virus diseases

Many of the world's most important infectious diseases are transmitted to humans by insect vectors. Attempts to eliminate the insect vectors or to immunize and protect the human host have thus far been unsuccessful. Alteration of insect vector competence is an alternative strategy to interrupt the cycle of disease transmission. Preliminary data from the Principal Investigator's laboratory document the unique capability of retroviral vectors containing the envelope glycoprotein of vesicular stomatitis virus (VSV-G) to infect and stably integrate into a broad range of non-mammalian cells including fish, frog, newt, and insect cells. These pantropic, replication-incompetent vectors have been used to create transgenic fish with stable, Mendelian segregation of the provirus in the F2 generation. The collaborating investigators have demonstrated that microinjection of mosquito eggs with concentrated viral stocks results in a high percentage of adult transformants containing the provirus. Using a Sindbis vector expression system, the collaborating investigators have shown protection of mosquito cells expressing the nucleocapsid gene of flaviviruses from superinfection with the homologous viral strain. These data suggest that pantropic retroviral vectors can be used to genetically modify insects to prevent transmission of important human pathogens. The proposed project will bring together three different investigators with established expertise in mosquito vector biology, virology, and retroviral vector design with the following goals: 1) to develop VSV-G pseudotyped retroviral vectors that mediate stable gene expression in mosquito cell lines and in transgenic progeny of germline transformants and 2) to create transgenic mosquitoes with a pantropic retroviral vector expressing the nucleocapsid gene of different flaviviruses, to confer cellular resistance to superinfection following challenge with wild type virus. The current proposal is a unique opportunity to develop a transformation system for the mosquito vector community that will permit the testing of strategies to alter mosquito vector competence to prevent vector-borne disease transmission.

世界上许多最重要的传染病都传播到 人类通过昆虫媒介。尝试消除昆虫媒介或 迄今为止，免疫和保护人类宿主尚未成功。 改变昆虫媒介能力是一种替代策略 中断疾病传播周期。初步数据来自 首席研究员的实验室记录了独特的能力 含有囊泡包膜糖蛋白的逆转录病毒载体 口腔炎病毒（VSV-G）可广泛感染并稳定整合 非哺乳动物细胞，包括鱼、青蛙、蝾螈和昆虫细胞。这些 泛向性、无复制能力的载体已被用来创建 转基因鱼中原病毒具有稳定的孟德尔分离 F2代。合作研究人员已经证明 用浓缩病毒储备显微注射蚊子卵会导致 含有原病毒的成体转化体的比例很高。使用 Sindbis载体表达系统，合作研究人员 显示出对表达核衣壳基因的蚊子细胞的保护作用 来自同源病毒株重复感染的黄病毒。这些 数据表明，泛向逆转录病毒载体可用于遗传 改造昆虫以防止重要的人类病原体传播。 拟议的项目将汇集三名不同的研究人员 拥有蚊媒生物学、病毒学等方面的专业知识 逆转录病毒载体设计具有以下目标： 1) 开发介导稳定的VSV-G假型逆转录病毒载体 蚊子细胞系和转基因后代中的基因表达 种系转化体和 2）用泛向逆转录病毒载体创建转基因蚊子 表达不同黄病毒的核衣壳基因，赋予 野生型攻击后细胞对重复感染的抵抗力 病毒。 当前的提案是发展转型的独特机会 蚊媒群落系统，将允许测试 改变蚊媒能力以预防媒介传播的策略 疾病传播。