HOMOLOGOUS GENE TRANSFER SYSTEMS FOR THE MOSQUITO

我们的长期目标是了解基因的调控。 埃及伊蚊在蚊子中的表达及其发育基因 这种昆虫的变形。在之前的授权期内，我们 启动了对两个20-羟基苯甲酸的分子结构的研究 羟基蜕皮激素诱导基因：卵黄蛋白原A1基因，它是 在脂肪体中表达，以及一个可能的卵黄膜蛋白基因 它在卵巢的滤泡细胞中表达。我们的注意力已经得到了 重点是识别这些基因的调控序列并开发 从这些序列中，嵌合结构可以表达为 体外培养转染伊蚊细胞。 我们已经确定了培养细胞中20-羟基蜕皮激素的反应。 来自白纹伊蚊和埃及伊蚊。20-的鉴定 培养蚊子细胞表达的羟基蜕皮激素诱导蛋白 支持该系统的持续发展。我们还开发了 在转基因细胞中表达基因以响应 蜕皮激素。最后，我们在培养的细胞中鉴定出一种25 kDa的蛋白质 其合成是由保幼激素类似物甲基丁二烯诱导的，以及 甲氧基丁二烯预适应后应用20-羟基蜕皮酮的增强作用 治疗。 我们建议通过以下方式来扩展这些研究：1)继续主要序列 20-羟基蜕皮激素诱导的卵黄蛋白原Al和卵黄素的分析 膜蛋白基因；2)检测染色质结构是否改变 发生在这些基因中，在能力的发展过程中， 对激素的刺激；3)建立了一种体外转录检测方法 蚊子基因的分析；4)继续开发同源基因 这些基因在转基因细胞中表达的转化系统 培养的细胞。 考虑到蚊子的基因改造是一个合理的目标 重组DNA技术提供的强大工具，以及快速 在其他有机体的转化方面取得了进展。给定 蚊子的医学价值显而易见，选择这种昆虫作为 基因转化的一个特定目标是显而易见的。

Our long term goals are directed towards understanding regulation of gene expression in the mosquito, Aedes aegypti, and developing genetic transformation of this insect. During the previous grant period we initiated an investigation of the molecular structure of two 20- hydroxyecdysone-inducible genes: the vitellogenin A1 gene, which is expressed in the fat body, and a putative vitelline membrane protein gene which is expressed in the follicle cells of the ovary. Our attention has focused on identifying regulatory sequences of these genes and developing from these sequences, chimeric constructs that can be expressed in transfected Aedes cells in culture. We have characterized the 20-hydroxyecdysone response in cultured cells from Aedes albopictus and Aedes aegypti. The identification of 20- hydroxyecdysone-inducible proteins expressed by cultured mosquito cells supports continued development of this system. We have also developed technology for expressing genes in transfected cells in response to ecdysone. Finally, we have identified in cultured cells a 25 kDa protein whose synthesis is induced by the juvenile hormone analog methoprene, and enhanced by treatment with 20-hydroxyecdysone following methoprene pre- treatment. We propose to extend these studies by: 1) continuing the primary sequence analysis of the 20-hydroxyecdysone-inducible vitellogenin Al and vitelline membrane protein genes; 2) testing whether changes in chromatin structure occur in these genes during the development of competence, and in response to hormone stimulation; 3) developing an in vitro transcription assay for analysis of the mosquito genes; 4) continuing to develop homologous transformation systems for expression of these genes in transfected cultured cells. Genetic transformation of mosquitoes is a reasonable goal given the powerful tools made available by recombinant DNA technology, and the rapid advances being made in the transformation of other organisms. Given the obvious medical importance of the mosquito, the choice of this insect as a particular target for genetic transformation is apparent.