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

描述(申请人提供)：我们工作的长期目标是开发控制蚊媒疾病传播的遗传方法。关键的技术进步将加速实现这一目标的进展，我们建议开发方法，加强对蚊子的分子分析，并减轻维持大量不同蚊子群体的负担。极细胞(PC)是发育中的双翅目昆虫胚胎形成的第一批生殖系细胞，是卵子和精子的祖细胞。目前用于蚊子转化的PC靶向基因转移技术还没有达到果蝇的效率水平，而且持续维护蚊子品系需要大量的人力和成本。我们在此建议分离可存活的PC，以开发创新的方法来应对蚊子转化的挑战。分离的PC将作为试剂，用于研究蚊子原始生殖细胞的体外利用、长期保存和维护的新技术。此外，对分离的PC进行转录组分析将为蚊子生殖系的发育提供新的见解，并增加有助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一种荧光蛋白，从而能够分离出具有活力的蚊子PC作为试剂，用于许多应用。其具体目的是：1)构建和检测含有生殖系特异性Nanos同源基因调控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中的表达；2)通过荧光激活细胞分选(FACS)分离EGFP标记的PC；3)通过胚胎移植和生殖系嵌合体的确认来检测PC的活性。与公共卫生有关的蚊子是引起寄生虫病和病毒性疾病的病原体的重要媒介。基因控制策略受到低效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。孤立的极地电池提供了制定减轻这些挑战的程序的机会。

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.