Pole cell isolation from the mosquitoes, Aedes aegypti and Anopheles stephensi

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DESCRIPTION (provided by applicant): The long-term objective of our work is to develop genetic approaches to controlling transmission of mosquito-borne diseases. Key technical advances will accelerate progress towards this goal, and we propose to develop methods that enhance molecular analyses of mosquitoes as well as alleviate the burden of maintaining large numbers of different mosquito colonies. Pole cells (PC) are the first germline cells formed in the developing dipteran embryo and are progenitors for eggs and sperm. PC-targeted gene transfer technologies used currently for mosquito transformation have yet to achieve the levels of efficiency attained with fruit flies, and continuous maintenance of mosquito strains requires much labor and cost. We propose here the isolation of viable PC for developing innovative approaches to the challenges of mosquito transformation. Isolated PC will serve as reagents for investigation of novel techniques for the utilization, long term- storage and maintenance of mosquito primordial germ cells, ex vivo. Additionally transcriptome analysis of isolated PC will yield new insights into mosquito germline development and increase the inventory of genes useful for the refinement of gene drive mechanisms. We propose to use mosquito nanos control sequences to express a fluorescent protein that would enable isolation of viable mosquito PC as reagents for a number of applications. The Specific Aims are to: 1) Construct and test modified transposable elements containing the regulatory DNA sequences of germline-specific nanos orthologous genes to drive the expression of the fluorescent reporter gene EGFP in the PC of Aedes aegypti and Anopheles stephensi; 2) Isolate EGFP-tagged PC by Fluorescence Activated Cell Sorting (FACS); 3) Test PC viability by transplantation into embryos and confirmation of germline mosaics. PUBLIC HEALTH RELEVANCE Mosquitoes are important vectors of the pathogens that cause parasitic and viral diseases. Genetic control strategies are hampered by inefficient transgenesis technologies and the labor-intensive requirements for maintaining large numbers of mosquito strains. Isolated pole cells offer the opportunity to develop procedures to mitigate these challenges.