Genetic tools for manipulation of mosquitoes

描述(申请人提供)：蚊媒传播的病原体给人类带来巨大的痛苦。媒介蚊子的基因组序列是了解和利用媒介特有的过程以减少疾病负担的重要资源。然而，对这些过程的研究的一个主要限制是缺乏可用的遗传工具来操纵体内的基因表达。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新方法将DNA输送到胚胎的可行性。我们的研究记录了从埃及伊蚊和冈比亚按蚊的卵子到成虫阶段的持续的质粒DNA和报告基因的活性，在将质粒传递给处于阶段的雌性蚊虫之后。我们现在建议在这一背景下，测试“垂直递送方法”的潜在应用。在目标1中，我们将优化方法，并确定报告基因的瞬时表达是否可以用于鉴定卵巢和中肠的成年雌性特异性启动子。在目标2中，我们将确定是否可以使用转座元件构建的垂直递送来有效地产生Ae的转基因系。埃及斑潜蝇和An.冈比亚亚目。这些目标的成功实现将提供新的方法，极大地促进未来对蚊子生物学的分析。 公共卫生相关性：控制蚊子和减少病媒传播疾病负担的公共卫生战略的相关性包括释放携带主要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。我们拟议的研究将对所有这些地区以及任何其他受益于分析蚊子基因功能的能力的地区产生影响。高效的转基因蚊子的产生和瞬时基因表达分析的新方法将对基本的生物学问题以及疾病控制的应用方法产生重大和持久的影响。。

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. .