Molecular genetic control of the immature female mosquito vector

描述（由申请人提供）：对于世界上超过40%的生活在蚊媒疾病流行地区的人以及新兴新地区的人来说，由雌蚊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新的解决方案来控制媒介和病原体传播。为了应对这一挑战，我们建议以专性疾病载体--雌蚊为目标，并采用一种新的方法，在雌蚊成为病原体载体之前的不成熟阶段消除雌蚊。最近，我们分离，从一个伊蚊atropalpus六聚体基因（Hex-1.2），特定的增强子启动子调控DNA序列，我们称之为六聚体增强子（Hex-Enh）。Hex-Enh靶向雌性和关键昆虫组织脂肪体（或肝脏）的基因活性，并且主要在晚期幼虫和早期蛹中有活性，即，未成熟的雌性蚊子因此，我们假设Hex-Enh构成了一个转录调节模块，可以驱动基因表达来破坏雌性脂肪体并导致未成熟雌性蚊子立即死亡。我们将通过以下具体目的在冈比亚按蚊和埃及伊蚊中检验这一假设。目的1：阻断和杀死未成熟的雌性Ae。通过使用Hex-Enh将细胞死亡基因（Michelob_x）靶向表达到雌性幼虫和蛹脂肪体中，我们将产生转基因Ae。埃及伊蚊携带1）在Hex-Enh控制下的四环素抑制性转录激活因子基因（tTAV）和2）作为效应基因的Michelob_x基因，其仅在雌性幼虫/蛹中被tTAV诱导。使用这种为蚊子设计的诱导性“tet-off”系统，我们期望通过消融脂肪体来选择性地杀死雌性蚊子幼虫和蛹。目的2：阻止发育，杀死未成熟的雌性安。通过使用Hex-Enh将归巢核酸内切酶I-PpoI的表达靶向雌性幼虫和蛹脂肪体，预测I-PpoI表达会“撕碎”X染色体，导致脂肪体细胞死亡。我们将产生转基因的An。冈比亚，其中I-PpoI活性由Hex-Enh指导，在雌性幼虫和蛹中应导致脂肪体消融和雌性成虫羽化前死亡。到目前为止，还没有转基因技术可以作为一个女性特定的杀蚊幼虫剂。我们提出的杀死未成熟的雌性蚊子可以应用于改善两个重要的蚊子控制策略：不育昆虫技术（SIT），通过开发新的遗传性别鉴定菌株和释放只有男性;和“释放昆虫携带显性致死”（RIDL），通过释放男性携带女性特定的显性致死作用在最有效的成虫前阶段。如果成功的话，我们提出的控制这两种主要蚊子的新技术的发展将大大减少世界范围内由按蚊或库蚊传播的多种传染病的传播。

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.