Expression of highly controllable genes in insect cells using a modified tetracycline-regulated gene expression system
Expression of highly controllable genes in insect cells using a modified tetracycline-regulated gene expression system
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DOI：
10.1016/s0168-1656(00)00247-9
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发表时间：
2000-06-09
影响因子：
4.1
通讯作者：
Chao, YC
中科院分区：
文献类型：
--
作者：
Wu, TY;Liono, L;Chao, YC
建立了一种用于昆虫细胞的改良四环素反应表达系统(TRES)。TRES包含两个组分：一个编码四环素可控的反式激活因子(TTA)，另一个包含驱动荧光素酶基因的TET操纵子DNA序列。我们的结果表明，人巨细胞病毒(CMV)启动子在昆虫细胞中不起作用，而人巨细胞病毒(CMV)启动子是在哺乳动物系统中强表达TTA所必需的。因此，需要进一步修改。利用链球绦虫多核型多角体病毒(AcMNPV)的p10启动子在Sf9、Sf21和Tn368细胞中高效表达TTA。当两个组分在修饰的TRES中被共转染时，这些细胞中的荧光素酶活性被检测到高达258倍。为了进一步简化实验，将p10启动子驱动的TTA插入到AcMNPV中。TTA表达重组病毒感染含有第二TRES组分的表达荧光素酶的重组病毒后，荧光素酶活性也明显增强。四环素显著抑制了这些系统中荧光素酶的表达，无论是在质粒中还是在病毒中的TTA基因和在质粒中的荧光素酶的表达。进一步研究了四环素对TRES作用的时程动力学。在50h内，对应于四环素的添加或去除，荧光素酶活性分别被完全抑制或激活。这些实验为进一步在昆虫细胞中广泛应用分子生物学研究建立了一个调控良好的基因表达系统。(C)2000由爱思唯尔科学公司出版。保留所有权利。
A modified tetracycline-responsive expression system (TRES) for use in insect cells was developed. The TRES contains two components: one encodes a tetracycline-controllable transactivator (tTA) and the other contains a tet operator DNA sequence to drive the luciferase gene. Our results show that the human cytomegalovirus (CMV) promoter, an essential part for strong tTA expression in mammalian system, was not functional in insect cells. Thus further modifications were required. Functional tTA was efficiently expressed hi Sf9, Sf21, and TN368 cells by the p10 promoter of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) in plasmid form with virus co-infection. An increase of up to 258-fold of luciferase activity was detected in these cells when both components in modified TRES were co-transfected. In order to further simplify the experiment, tTA, which is driven by the p10 promoter, was inserted into AcMNPV. Luciferase activity was also strongly stimulated by the infection of this tTA expression-recombinant virus with the transfection of a plasmid containing the second TRES component expressing luciferase. The luciferase expressions in these systems, either in plasmids or the tTA gene in virus and luciferase in plasmid, were significantly suppressed by tetracycline. The time course kinetics of tetracycline action to the TRES were further studied. Within a time span of 50 h, the luciferase activities could be fully suppressed or activated, respectively, corresponding to the addition or removal of tetracycline. These experiments have established a well-regulated gene expression system for further broad applications of molecular biological studies in insect cells. (C) 2000 Published by Elsevier Science B.V. All rights reserved.