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。通过更好地了解这些昆虫的遗传学和生理学，可以产生控制蚊子媒介的替代策略。通过开发有效和可重复的转基因操作手段，将大大促进蚊子遗传研究的进展。这种能力的一个关键组成部分是鉴定和操纵昆虫转座子，这些转座子能够有效地将基因导入蚊子基因组，允许基因标记，并保持基因处于活性状态。这一领域研究的融合已经成功地对蚊子进行了几种昆虫转座子的转基因操作，每个转座子都有自己独特的性质。昆虫转座子piggyBac已被证明对包括蚊子在内的多种昆虫物种的遗传操纵是有效的。最近，我们利用朱砂眼颜色标记基因证明了埃及伊蚊与piggyBac的转化。该提案寻求大量资金，以了解和利用piggyBac转座子建立和维持伊丁蚊种转基因菌株的能力。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，将构建含有所需基因的其他基于piggyback的转基因载体，进行测试，并与可选择的转基因标记结合。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，并使用多种技术进行分析，包括southern杂交，PCR扩增和测序，Northern和Western分析，以及免疫荧光（视情况而定）。转基因序列在蚊子基因组内的稳定性和运动的遗传特征将在几代内进行检查。该研究将为加强蚊子遗传学和生理学的基础研究提供一个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.