Shotkoski F A, Fallon A M
Department of Entomology, University of Minnesota, St. Paul.
Am J Trop Med Hyg. 1994 Apr;50(4):433-9. doi: 10.4269/ajtmh.1994.50.433.
Novel approaches to control of vector-borne disease include potential use of transgenic insects, in which molecular mechanisms will be induced to prevent transmission of pathogenic organisms. The infrastructure essential to this technology includes the cloning of essential genes from vector insects, and the development of efficient transformation strategies. In this study, we use a continuous mosquito (Aedes albopictus) cell line and a cloned mosquito dihydrofolate reductase gene to demonstrate a transgenic approach that may be used to select for the presence or absence of particular gene functions in transfected cells. Plasmids containing the dihydrofolate reductase gene in sense and antisense orientation, under the regulation of a temperature-inducible promoter, were expressed in stably transfected mosquito cells. At the normal growth temperature of 28 degrees C, or after mild heat induction at 34 degrees C, expression of the dihydrofolate reductase construct in sense orientation had little effect on cell growth. In contrast, recovery of clones transfected with the antisense construct was reduced, and induction of antisense transcripts at 34 degrees C further compromised cell growth and viability. Clones transfected with the sense construct retained significantly higher copy numbers of foreign DNA than did cells transfected with the antisense construct. These studies provide a basis for use of sense and antisense dihydrofolate reductase constructs to recover transfected mosquito cells with specific desired phenotypes, based on the relative expression of cloned genes of interest.
控制媒介传播疾病的新方法包括潜在地使用转基因昆虫，其中将诱导分子机制来预防病原生物的传播。这项技术所需的基础设施包括从媒介昆虫中克隆必需基因，以及开发高效的转化策略。在本研究中，我们使用一种连续的蚊子（白纹伊蚊）细胞系和一个克隆的蚊子二氢叶酸还原酶基因来展示一种转基因方法，该方法可用于选择转染细胞中特定基因功能的存在与否。含有在温度诱导型启动子调控下的正向和反向二氢叶酸还原酶基因的质粒，在稳定转染的蚊子细胞中表达。在正常生长温度28℃时，或在34℃轻度热诱导后，正向二氢叶酸还原酶构建体的表达对细胞生长影响很小。相比之下，用反向构建体转染的克隆的回收率降低，并且在34℃诱导反义转录本进一步损害细胞生长和活力。用正向构建体转染的克隆比用反向构建体转染的细胞保留了显著更高拷贝数的外源DNA。这些研究为基于感兴趣的克隆基因的相对表达，使用正向和反向二氢叶酸还原酶构建体来筛选具有特定所需表型的转染蚊子细胞提供了基础。
