Genetic tools for manipulation of mosquitoes

描述（由申请人提供）：蚊媒病原体造成巨大的人类痛苦。病媒蚊子的基因组序列是了解和利用病媒特异性过程以减少疾病负担的重要资源。然而，这些过程的研究的一个主要限制是现有的遗传工具在体内操纵基因表达的不足。我们的初步研究证明了一种基于雌性蚊子生物学关键要素的DNA递送到胚胎的新方法的可行性。我们的研究文件的持久性的质粒DNA和报告基因的活性，从鸡蛋到成人阶段的埃及伊蚊和冈比亚按蚊，交付后的阶段女性的质粒。我们现在建议在这一背景的基础上，测试“垂直交付方法”的潜在应用。在目标1中，我们将优化的方法，并确定瞬时表达的报告基因是否可以用来确定成年女性特异性卵巢和中肠启动子。在目标2中，我们将确定是否可以使用转座因子构建体的垂直递送来有效地产生Ae的转基因系。aegypti和An.冈比亚。这些目标的成功完成将提供新的方法，将大大促进未来的蚊子生物学分析。 公共卫生相关性：与控制蚊子和减少病媒传播疾病负担的公共卫生战略相关的措施包括释放携带显性致死基因或抗病原体机制的转基因蚊子。该领域的重要方向还包括嗅觉通路的解剖，杀虫剂抗性的机制，以及与人类叮咬相关的关键行为的进化和生态学。我们提出的研究将对所有这些领域以及任何其他受益于分析蚊子基因功能的领域产生影响。转基因蚊子的高效产生和基因表达瞬时分析的新方法将对基本生物学问题以及疾病控制的应用方法产生重大而持久的影响。.

DESCRIPTION (provided by applicant): Mosquito-borne pathogens cause enormous human suffering. The genome sequences of vector mosquitoes are important resources for understanding and exploitation of vector-specific processes to reduce the burden of disease. However, a major limitation to studies of these processes is the inadequacy of available genetic tools for manipulating gene expression in vivo. Our preliminary studies demonstrate the feasibility of a new method for DNA delivery to embryos based on key elements of female mosquito biology. Our studies document the persistence of plasmid DNA and reporter gene activity from the egg to the adult stage in both Aedes aegypti and Anopheles gambiae, following delivery of the plasmids to the staged females. We now propose to build on this background and test potential applications of the "vertical delivery method". In Aim 1, we will optimize the method and determine whether transient expression of reporter genes can be used to identify adult female-specific promoters for the ovary and midgut. In Aim 2, we will determine whether vertical delivery of transposable element constructs can be used to efficiently generate transgenic lines of Ae. aegypti and An. gambiae. Successful completion of these aims will provide new methods that will greatly facilitate future analyses of mosquito biology. PUBLIC HEALTH RELEVANCE: Relevance to public health strategies for controlling mosquitoes and reducing the burden of vector borne disease include the release of transgenic mosquitoes that carry dominant lethal genes or anti- pathogen mechanisms. Important directions for the field also include the dissection of the olfactory pathways, the mechanisms of insecticide resistance, and the evolution and ecology of key behaviors associated with human biting. Our proposed study will have impacts on all of these areas and any others that benefit from the ability to analyze gene function within mosquitoes. Highly efficient generation of transgenic mosquitoes and new methods for transient analysis of gene expression will have significant and lasting impacts on both basic biological questions as well as applied approaches to disease control. .