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

描述（由申请人提供）：我们工作的长期目标是开发遗传方法来控制蚊媒疾病的传播。关键技术进步将加速实现这一目标的进展，我们建议开发增强蚊子分子分析并减轻维持大量不同蚊子群落负担的方法。极细胞（PC）是在双翅目胚胎发育过程中形成的第一个生殖细胞，是卵子和精子的祖细胞。目前用于蚊子转化的PC靶向基因转移技术尚未达到果蝇所达到的效率水平，并且蚊子品系的持续维护需要大量的劳动力和成本。我们在此建议分离可行的 PC，以开发创新方法来应对蚊子转化的挑战。分离的PC将作为试剂用于研究离体蚊子原始生殖细胞的利用、长期储存和维持的新技术。此外，分离 PC 的转录组分析将为蚊子种系发育提供新的见解，并增加可用于完善基因驱动机制的基因库存。我们建议使用蚊子纳米控制序列来表达荧光蛋白，从而能够分离出可行的蚊子 PC 作为许多应用的试剂。具体目标是： 1）构建并测试含有种系特异性纳米直系同源基因的调控DNA序列的修饰转座元件，以驱动荧光报告基因EGFP在埃及伊蚊和斯氏按蚊的PC中表达； 2）通过荧光激活细胞分选（FACS）分离EGFP标记的PC； 3) 通过移植到胚胎中并确认种系嵌合体来测试 PC 的活力。公共卫生相关性 蚊子是引起寄生虫病和病毒性疾病的病原体的重要媒介。基因控制策略受到效率低下的转基因技术和维持大量蚊子品系的劳动密集型要求的阻碍。孤立的极电池提供了开发程序来缓解这些挑战的机会。

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.