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

从蚊子、埃及伊蚊和史氏按蚊中分离杆细胞

• 批准号: 7620412
• 负责人: Anthony A. James
• 金额: $ 19.11万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7620412
• 关键词: Aedes; Anopheles Genus; Cell Separation; Cell Survival; Cells; Culicidae; DNA; DNA Sequence; DNA Transposable Elements; Development; Drosophila genus; Embryo; Equipment and supply inventories; Fluorescence-Activated Cell Sorting; Gene Expression Profile; Gene Transfer; Gene Transfer Techniques; Genes; Genetic; Goals; Insecta; Investigation; Maintenance; Methods; Molecular; Mosquito-borne infectious disease; Orthologous Gene; Procedures; Proteins; Reagent; Reporter Genes; Structure of primordial sex cell; Techniques; Technology; Testing; Transplantation; Transposase; Virus Diseases; Work; cell transformation; cost; egg; innovation; insight; interest; novel; pathogen; progenitor; public health relevance; sperm cell; technology development; transmission process; vector; vector mosquito

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.

描述（由申请人提供）：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，我们建议开发增强蚊子分子分析的方法，并减轻维持大量不同蚊子群体的负担。极细胞（PC）是双翅目昆虫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的效率水平，并且蚊子品系的持续维持需要大量的劳动力和成本。我们建议在这里的隔离可行的PC开发创新的方法，蚊子转化的挑战。分离的原核细胞将作为研究离体利用、长期保存和维持蚊子原始生殖细胞的新技术的试剂。此外，分离的PC的转录组分析将产生对蚊子生殖系发育的新见解，并增加对基因驱动机制的完善有用的基因的库存。我们建议使用蚊子nanos控制序列来表达荧光蛋白，这将使可行的蚊子PC作为试剂的分离用于许多应用。具体目标是：1）构建并测试含有种系特异性nanos orthotropic基因的调控DNA序列的修饰的转座元件，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）通过移植到胚胎中并确认种系嵌合体来测试PC活力。蚊子是引起寄生虫病和病毒病的病原体的重要媒介。遗传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。孤立极细胞提供了机会，以制定程序，以减轻这些挑战。