Pfeifer T A, Hegedus D D, Grigliatti T A, Theilmann D A
Department of Zoology, University of British Columbia, Vancouver, Canada.
Gene. 1997 Apr 1;188(2):183-90. doi: 10.1016/s0378-1119(96)00756-1.
The antibiotic Zeocin, a derivative of phleomycin, was evaluated for use as a selection system in both dipteran and lepidopteran insect cell lines. Growth of Drosophila cell lines, Kc1 and SL2, was inhibited at Zeocin concentrations of 50 and 75 microg/ml, respectively, while the Spodoptera cell line, Sf9, was inhibited at a concentration of 250 microg/ml Zeocin. The mammalian cytomegalovirus (CMV) and Simian virus 40 (SV40) early promoters did not function in these insect cell lines. Several baculovirus-derived immediate-early (IE) promoters from the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) and Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were used to drive expression of the Zeocin resistance gene (ble) in these cell lines. The resulting plasmid vectors enabled selection of Zeocin-resistant cell lines within 3-4 weeks. Gene amplification events in the presence of increasing Zeocin concentrations were not detected using Southern blot analysis. Furthermore, the function of the baculovirus IE promoters, as demonstrated by beta-galactosidase expression, was not detectable in a variety of mammalian cell lines tested. A cloning/shuttle vector, containing ten unique restriction sites, was constructed which allows for selection of Zeocin resistance in insect cell lines and in Escherichia coli.
抗生素博来霉素的衍生物杀稻瘟菌素，被评估用作双翅目和鳞翅目昆虫细胞系的筛选系统。果蝇细胞系Kc1和SL2在杀稻瘟菌素浓度分别为50和75微克/毫升时生长受到抑制，而草地贪夜蛾细胞系Sf9在杀稻瘟菌素浓度为250微克/毫升时生长受到抑制。哺乳动物巨细胞病毒（CMV）和猿猴病毒40（SV40）早期启动子在这些昆虫细胞系中不起作用。来自太平洋折翅蛾多粒包埋核型多角体病毒（OpMNPV）和苜蓿银纹夜蛾多粒包埋核型多角体病毒（AcMNPV）的几种杆状病毒衍生的立即早期（IE）启动子被用于驱动这些细胞系中杀稻瘟菌素抗性基因（ble）的表达。所得质粒载体能够在3至4周内筛选出抗杀稻瘟菌素的细胞系。使用Southern印迹分析未检测到在杀稻瘟菌素浓度增加时的基因扩增事件。此外，通过β-半乳糖苷酶表达证明的杆状病毒IE启动子的功能，在所测试的多种哺乳动物细胞系中未检测到。构建了一种含有十个独特限制酶切位点的克隆/穿梭载体，其允许在昆虫细胞系和大肠杆菌中筛选杀稻瘟菌素抗性。
