HOMOLOGOUS GENE TRANSFER SYSTEMS FOR THE MOSQUITO

我们的长期目标是了解基因调控 表达在蚊子，埃及伊蚊，和发展中的遗传 这种昆虫的变化。在上一个资助期内，我们 开始了对两个20- 羟基蜕皮素诱导基因：卵黄蛋白原A1基因， 在脂肪体中表达，和一个假定的卵黄膜蛋白基因 其在卵巢的卵泡细胞中表达。我们的注意力 专注于确定这些基因的调控序列， 从这些序列中，可以表达嵌合构建体， 转染的伊蚊细胞培养。 我们的特点是20-羟基蜕皮激素的反应，在培养细胞 来自白纹伊蚊和埃及伊蚊。识别20- 蚊细胞表达的羟基蜕皮素诱导蛋白 支持该系统的持续发展。我们还开发了 在转染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了一种25 kDa的蛋白质， 其合成由保幼激素类似物甲氧普烯诱导，和 加强与20-羟基蜕皮激素治疗后， 治疗 我们建议扩展这些研究：1）继续初级序列 20-羟基蜕皮激素诱导的卵黄蛋白原Al和卵黄蛋白的分析 膜蛋白基因; 2）检测染色质结构是否发生变化 在这些基因中发生在能力的发展过程中， 激素刺激; 3）开发体外转录测定， 蚊子基因的分析; 4）继续开发同源 用于在转染细胞中表达这些基因的转化系统 培养细胞 蚊子的遗传转化是一个合理的目标， 重组DNA技术提供了强大的工具， 在改造其他生物体方面取得的进展。鉴于 蚊子明显的医学重要性，选择这种昆虫作为 遗传转化的特定靶是显而易见的。

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.