Molecular genetic control of the immature female mosquito vector

描述（由申请人提供）：对于生活在蚊媒疾病流行地区以及新兴地区的40%以上的世界人口来说，由雌性蚊子传播的传染病继续对健康和生命构成威胁，并挑战研究人员寻找媒介控制和病原体传播的新解决方案。为了应对这一挑战，我们提出了一种新的方法，即在雌性蚊子成为病原体载体之前，将其消灭在未成熟阶段。最近，我们从atropalus伊蚊六聚氰胺基因（hexx -1.2）中分离出特异性的增强子-启动子调控DNA序列，我们将其命名为六聚氰胺增强子（hexx -enh）。Hex-Enh针对的是雌性和关键昆虫组织脂肪体（或肝脏）的基因活性，主要在幼虫晚期和早期蛹（即未成熟的雌蚊）中活跃。因此，我们假设Hex-Enh构成了一个转录调控模块，可以驱动基因表达破坏雌性脂肪体，导致未成熟雌蚊立即死亡。我们将通过以下具体目标在冈比亚按蚊和埃及伊蚊中验证这一假设。目的1：阻断发育并杀死未成熟的雌性伊蚊。利用Hex-Enh靶向表达细胞死亡基因（Michelob_x）到雌性幼虫和蛹脂肪体。我们将产生转基因Ae。携带1)Hex-Enh控制的四环素抑制转录激活因子基因（tTAV）和2)Michelob_x基因作为tTAV在雌性幼虫/蛹中单独诱导的效应基因。利用这种为蚊子设计的诱导“释放”系统，我们期望通过烧蚀脂肪体来选择性地杀死雌蚊幼虫和蛹。目的2：阻止发育，并杀死未成熟的雌性安。利用hexx - enh靶向冈比亚蚊雌幼虫和蛹脂肪体的归巢酶I-PpoI的表达。预计I-PpoI的表达会“撕碎”X染色体，导致脂肪体细胞死亡。我们将产生转基因安。在雌性幼虫和蛹中，由Hex-Enh引导的I-PpoI活性应导致脂肪体消融和雌性在成虫羽化前死亡。到目前为止，还没有转基因技术可以作为雌性蚊子特有的杀幼虫剂。我们提出的杀死未成熟雌蚊可用于改善两种重要的蚊虫控制策略：不育昆虫技术（Sterile Insect Technique， SIT），通过开发新的遗传性别株系和只释放雄蚊；以及“释放携带显性致死行为的昆虫”（RIDL），即在最有效的成虫前阶段释放携带雌性特有显性致死行为的雄性昆虫。如果成功，我们提出的控制这两种主要蚊子的新技术将大大减少由按蚊或烹饪蚊子传播的多种传染病在世界范围内的传播。

DESCRIPTION (provided by applicant): For the more than 40% of the world's people living in areas endemic for mosquito-borne diseases-as well as for those in emerging new areas-infectious diseases vectored by female mosquitoes continue to pose threats to health and life and to challenge researchers to find novel solutions to vector control and pathogen transmission. To meet this challenge, we propose to target the obligatory disease vector, the female mosquito, and to do so in a novel approach that eliminates the female mosquito at an immature stage before it can become a pathogen vector. Recently, we isolated, from an Aedes atropalpus hexamerin gene (Hex-1.2), specific enhancer-promoter regulatory DNA sequences, which we have termed the hexamerin-enhancer (Hex-Enh). The Hex-Enh targets gene activity both uniquely in females and to a critical insect tissue, the fat body (or liver), and is active primarily in late larvae and early pupae, i.e., immature, female mosquitoes. Thus, we hypothesize that the Hex-Enh constitutes a transcriptional regulatory module that can drive gene expression to destroy the female fat body and cause immediate death of immature female mosquitoes. We will test this hypothesis in Anopheles gambiae and Aedes aegypti by the following Specific Aims. Aim 1: Block development of, and kill, the immature female Ae. aegypti mosquito by using the Hex-Enh to target expression of a cell death gene (Michelob_x) to the female larval and pupal fat body. We will generate transgenic Ae. aegypti carrying 1) the tetracycline-repressible transcriptional activator gene (tTAV) under control of the Hex-Enh and 2) the Michelob_x gene as an effector gene to be induced exclusively in female larvae/pupae by tTAV. Using this inducible "tet-off" system engineered for mosquitoes, we expect to selectively kill female mosquito larvae and pupae by ablation of the fat body. Aim 2: Block development of, and kill, the immature female An. gambiae mosquito by using the Hex-Enh to target the expression of the homing endonuclease I-PpoI to the female larval and pupal fat body. I-PpoI expression is predicted to "shred" the X chromosome, resulting in the death of fat body cells. We will generate transgenic An. gambiae in which I-PpoI activity, directed by the Hex-Enh, in female larvae and pupae should result in ablation of the fat body and female death before adult eclosion. To date, no transgenic technology exists that can act as a female-specific mosquito larvicide. Our proposed killing of immature female mosquitoes can be applied to improve two important mosquito control strategies: Sterile Insect Technique (SIT), by developing novel genetic sexing strains and the release of only males; and "release of insects carrying a dominant lethal" (RIDL), by releasing males carrying a female-specific dominant lethal acting at the most effective preadult stage. If successful, our proposed development of novel technology for control of both major types of mosquitoes should dramatically reduce transmission of multiple infectious diseases vectored by either anopheline or culicine mosquitoes worldwide.