FLP-MEDIATED RECOMBINATION IN THE MOSQUITO AEDES AEGYPTI

点击翻译按钮获取中文摘要

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.