FLP-MEDIATED RECOMBINATION IN THE MOSQUITO AEDES AEGYPTI

病媒传播的寄生虫病仍然是对生命和 世界上许多地区的健康状况。我们宏伟的长期目标 是开发对载体进行分子遗传操作的方法 蚊子最终了解病媒与病原体的相互作用 为控制媒介传播疾病提供新的手段。一个 在这个方向上的重要一步是开发生成 转基因蚊子。这样的蚊子将提供一个系统，在这个系统中 基因操作对病原菌繁殖和传播的影响 可能会被评估。此外，这些菌株还可以用于检测 疾病控制协议，要求释放经基因改变的 蚊子。这项提案的具体目标是：1)确定最优 FLP-重组酶水平，在特定位点期间最大化整合 埃及伊蚊早期胚胎重组；2)建立埃及伊蚊品系 含有FLP-重组酶靶点FRT的转基因蚊子 目前的蚊子转化方案；3)评估和优化 FLP介导的外源基因整合与表达 重组成含有FRT的转基因蚊子。FLP- 重组酶mRNA将在体外合成并用于微量注射 蚊子胚胎。这将提供短暂和受控的 重组酶活性，应提供稳定的整合事件 蚊子。将制造携带FRT目标位置的蚊子品系 利用已经被发现的低频、非法重组 被证明发生在蚊子身上。这样的菌株应该是 FLP介导的高频重组的底物。的频率 FLP介导的整合到含有FRT的蚊子品系中将是 以及整合基因的表达水平。首次登记税- 含有具有最佳高频平衡的蚊子品系 整合和外源基因表达将成为 分析蚊子的基因表达。

Vector-borne parasitic diseases continue to be a major threat to life and health throughout many parts of the world. Our broad long term objective is to develop methods for molecular genetic manipulation of vector mosquitoes ultimately to understand vector-pathogen interactions and provide novel means for control of vector-transmitted disease. An important step in this direction is to develop methods for generating transgenic mosquitoes. Such mosquitoes would provide a system in which the effects of genetic manipulation on pathogen propagation and transmission could be evaluated. In addition, such strains could be used to test disease-control protocols that require the release of genetically-altered mosquitoes. The specific aims of this proposal are; 1) to define optimum FLP-recombinase levels that maximize integration during site-specific recombination in early Aedes aegypti embryos; 2) to establish lines of transgenic mosquitoes containing an FLP-recombinase target site, FRT, using current mosquito transformation protocols; 3) to evaluate and optimize the integration and expression of exogenous genes incorporated via FLP-mediated recombination into the FRT-containing transgenic mosquitoes. FLP- recombinase mRNA will be synthesized in vitro and used to microinject mosquito embryos. This will deliver a brief and controlled amount of recombinase activity that should provide for stable integration events in the mosquito. Strains of mosquitoes carrying FRT target sites will be made utilizing the low frequency, illegitimate recombination that has been demonstrated to take place in mosquitoes. Such strains should be substrates for high frequency FLP-mediated recombination. The frequency of FLP-mediated integration into the FRT-containing mosquito strains will be determined, as well as the expression levels of integrated genes. The FRT- containing mosquito strain that has the best balance of high-frequency integration and exogenous gene expression will become a standard strain for assaying gene expression in mosquitoes.