TRANSGENIC ENGINEERING OF AEDINE MOSQUITOES

蚊媒传染病继续对世界大部分地区的健康和经济产生重大影响。通过更好地了解蚊虫的遗传学和生理学，可以产生控制蚊媒的替代策略。通过开发有效和可重现的转基因操作手段，蚊子遗传研究的进展将得到极大的促进。这种能力的一个关键组成部分是识别和操纵昆虫转座子，这些转座子能够有效地将基因转移到蚊子基因组中，允许基因标记，并保持基因处于活跃状态。这一领域的研究汇聚在一起，成功地利用几个昆虫转座子对蚊子进行了转基因操作，每个转座子都有自己独特的特性。昆虫转座子iggyBac已经被证明在包括蚊子在内的广泛不同的昆虫物种中进行基因操作是有效的。最近，我们利用朱砂眼部着色标记基因，对埃及伊蚊进行了基因转化。这项提案寻求大量资金，以了解和利用iggyBac转座子建立和维护转基因伊蚊品种的能力。另外，还将构建、测试并与可选择的转基因标记相结合，在普遍存在的昆虫启动子或组织特异性蚊子启动子的控制下，包含所需基因的基于iggyBac的转基因载体。在G1代选择共转化标记基因后，将检测转座子和载体基因的存在和表达，并酌情使用各种技术进行分析，包括Southern杂交、PCR扩增和测序、Northern和Western分析以及免疫荧光。蚊子基因组内转基因序列稳定性和移动的遗传特征将在几代人的时间内进行检查。这项研究将为显着加强蚊子遗传学和生理学的基础研究工作提供令人兴奋和迫切需要的能力。

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 aegypti 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 G1 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.