Molecular genetic control of the immature female mosquito vector

描述(申请人提供)：对于生活在蚊媒疾病流行地区以及新兴新地区的超过40%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病媒控制和病原体传播的新解决方案。为了应对这一挑战，我们建议以强制性疾病媒介雌性蚊子为目标，并以一种新的方法这样做，在雌性蚊子成为病原体媒介之前，在未成熟阶段消灭雌性蚊子。最近，我们从黑斑伊蚊的六聚体蛋白基因(Hex-1.2)中分离到了特异的六聚体增强子-启动子调控DNA序列，我们称之为六聚体增强体(Hex-Enh)。Hex-Enh针对雌性和关键昆虫组织-脂肪体(或肝脏)的基因活性，主要作用于晚期幼虫和早期幼虫，即未成熟的雌性蚊子。因此，我们假设Hex-Enh构成了一个转录调控模块，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的立即死亡。我们将通过以下具体目标在冈比亚按蚊和埃及伊蚊中检验这一假说。目的1：阻断未成熟雌性Ae的发育并将其杀死。通过使用Hex-Enh将细胞死亡基因(Michelob_X)靶向表达到雌蚊幼虫和蛹脂肪体中。我们将产生转基因Ae。埃及斑潜蝇携带1)四环素可抑制转录激活基因(TTAV)在十六烷基腺病毒(Hex-Enh)控制下；2)Michelob_x基因作为效应基因由tTAV在雌性幼虫/蛹中唯一地诱导。利用这种为蚊子设计的可诱导的“tet-off”系统，我们希望通过消融脂肪身体来选择性地杀死蚊子的雌性幼虫和幼虫。目标2：阻止发育，并杀死未成熟的雌性AN。冈比亚蚊虫通过使用Hex-Enh来靶向表达归巢内切酶I-PpoI到雌幼虫和蛹脂肪体。据预测，I-PpoI的表达会“撕碎”X染色体，导致脂肪体细胞死亡。我们将产生转基因的AN。冈比亚亚种，在雌性幼虫和蛹中，由十六烷基硫酸酯(Hex-Enh)指导的I-PpoI活性应导致脂肪体消融，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女性特有的蚊子幼虫杀虫剂。我们建议的杀灭未成熟雌性蚊子可以用来改进两种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品系并只释放雄性蚊子；以及“释放携带显性致死基因的昆虫”(RIDL)，通过释放携带雌性特有显性致死蚊子的雄性蚊子，在最有效的成虫前阶段发挥作用。如果成功，我们建议开发的控制这两种主要蚊子的新技术应该会极大地减少由按蚊或库蚊传播的多种传染病在全球的传播。

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.