Transgenic engineering of Aedine mosquitoes

伊丁蚊的转基因工程

• 批准号: 6332193
• 负责人: Malcolm J. FRASER
• 金额: $ 22.1万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6332193
• 关键词: Aedes; arthropod genetics; biotechnology; genetic manipulation; genetically modified animals; transfection /expression vector; transposon /insertion element

DESCRIPTION (provided by applicant): Mosquito-borne infectious diseases continue to have significant impact on the health and economy of much of the world. Alternative strategies for control of mosquito vectors could arise through a greater understanding of the genetics and physiology of these insects. Progress in genetic research with mosquitoes will be greatly enhanced by the development of effective and reproducible means of transgenic manipulations. A key component of this capability is the identification and manipulation of insect transposons that are able to efficiently vector genes into the mosquito genome, permit gene tagging, and maintain genes in an active state. The convergence of research in this field has resulted in the successful transgenic manipulation of mosquitoes with several insect transposons, each with its own unique properties. The insect transposon piggyBac has been demonstrated to be effective for genetic manipulation in widely diverse insect species, including mosquitoes. Recently, we demonstrated transformation of Aedes acgypti with piggyBac using the cinnabar eye coloration marker gene. This proposal seeks significant funding to understand and exploit the capabilities of the piggyBac transposon for establishing and maintaining transgenic strains of Aedine mosquito species. Additional piggyBac-based transgene vectors containing desirable genes under the control of either ubiquitous insect promoters or tissue-specific mosquito promoters will be constructed, tested, and combined with the selectable markers for transgenesis. The presence and expression of the transposon and vectored genes will be detected following selection for the co-transforming marker gene at the Gi generation, and analyzed using a variety of techniques including Southern hybridization, PCR amplification and sequencing, Northern and Western analyses, and immunoflourescence, as appropriate. Genetic characterization of the stability and movement of transgene sequences within the mosquito genome will be examined over several generations. This research will provide an exciting and much needed capability for significant enhancement of the basic research effort in mosquito genetics and physiology.

描述（申请人提供）：蚊媒传染病 继续对许多国家的健康和经济产生重大影响， 世界可能出现控制蚊子媒介的替代战略 通过更好地了解这些基因和生理学， 昆虫用蚊子进行基因研究的进展将大大加强 通过开发有效的和可重复的转基因方法， 操纵这种能力的一个关键组成部分是识别和 操纵昆虫转座子， 进入蚊子基因组，允许基因标记，并保持基因在一个活跃的 状态在这一领域的研究的融合导致了成功的 用几种昆虫转座子对蚊子进行转基因操作， 有着自己独特的属性昆虫转座子piggyBac 被证明是有效的遗传操作在广泛不同的昆虫 物种，包括蚊子。最近，我们展示了 埃及伊蚊与piggyBac使用朱砂眼显色标记基因。这 该提案寻求大量资金来了解和利用这些能力， piggyBac转座子用于建立和维持转基因菌株 Aedine蚊子物种的。另外的基于piggyBac的转基因载体 含有受普遍存在的昆虫控制的理想基因 启动子或组织特异性蚊子启动子将被构建，测试， 并与选择标记组合用于转基因。的存在和 转座子和载体基因的表达将在以下检测 在Gi代选择共转化标记基因，和 使用多种技术进行分析，包括Southern杂交、PCR 扩增和测序，北方和西方分析，和 免疫荧光法，视情况而定。稳定性的遗传表征 转基因序列在蚊子基因组中的移动将被检查 持续了好几代这项研究将提供一个令人兴奋的， 大幅度加强基础研究工作所需的能力， 蚊子的遗传学和生理学。