Mosquitoes are major vectors of pathogens that cause devastating diseases which impact human health and national economies worldwide. The use of DNA transposons as tools for the genetic manipulation of mosquitoes holds great potential for the elucidation of pathogen-mosquito interactions. However, the current rates of transposon-mediated transformation of mosquitoes are suboptimal for functional genomics studies which are crucial to the development of novel genetic approaches to stem the increasing global incidence of mosquito-borne diseases, as such studies require the production of numerous transgenic lines in order to be effective. The occurrence of imprecise transposition events in which donor molecule flanking sequences are co-integrated with transposons is an additional concern for the production of transgenic mosquitoes. The long-term goal of this research is to increase the frequency of precise Mos1 mariner integrations into the germ line chromosomes of the mosquito, Aedes aegypti, a major vector of the Dengue, Yellow Fever and Chikungunya viruses. The hypothesis for this research is that an increase in the frequency of precise integrations of Mos1 into Ae. aegypti germ line chromosomes can be induced by altering the DNA and protein elements of Mos1. We propose the production of hyperactive Mos1 transposons by altering cis-acting transposition regulatory sequences identified through mutagenesis and deletion analysis. We further propose the evaluation of these mutant transposons using previously produced mutant Mos1 transposases in order to identify combinations of mutant transposons and transposases that display hyperactive rates of precise integrations into Ae. aegypti germ line chromosomes. Initial evaluations of mutant transposon donor constructs and mutant transposase helper constructs will be performed using plasmid-based assays in Ae. aegypti embryos, and final evaluations will be made based on data from mosquito germ line transformation experiments. Our approach will address both aspects of the long-term goal, as preliminary data indicate that increases in transposition frequency and precision can occur concurrently. The results of this research will make possible the efficient production of large numbers of Ae. aegypti transgenic lines for use in genetic studies of this medically important species. ? ? ?
