Jost M, Kari C, Rodeck U
The Wistar Institute of Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104, USA.
Nucleic Acids Res. 1997 Aug 1;25(15):3131-4. doi: 10.1093/nar/25.15.3131.
The recently introduced tetracycline (Tc)-regulatable eukaryotic gene expression system based on the Escherichia coli Tn 10 tetracycline operon has proven to be a powerful tool for controlled expression of a variety of genes in vitro as well as in vivo . Control elements of this expression system are contained in two separate plasmid vectors. The tTA vector encodes a transactivator protein and the tetP vector contains a responsive operator-promoter element (tetP) that controls gene expression depending on tTA binding. Establishment of cell lines expressing a gene of interest under tetP control requires two subsequent rounds of transfection and clonal selection after each transfection. Here we describe a modification of this system in which the tetP element is placed in an episomal EBNA-based plasmid that can be stably maintained in primate but not in rodent cells. Using HeLa and human melanoma cells, we show that upon transient or stable transfection a reporter gene is expressed in a Tc-regulated manner similar to the original system. Thus, this expression system combines the advantages of episomal vectors, such as high efficiency of transfection and time-efficient selection of mass cultures, with tight control of gene expression provided by the Tc-regulatable system.
最近引入的基于大肠杆菌Tn10四环素操纵子的四环素（Tc）调控真核基因表达系统，已被证明是一种在体外和体内控制多种基因表达的强大工具。该表达系统的控制元件包含在两个单独的质粒载体中。tTA载体编码一种反式激活蛋白，tetP载体包含一个响应性操纵子 - 启动子元件（tetP），它根据tTA的结合来控制基因表达。建立在tetP控制下表达感兴趣基因的细胞系需要两轮后续转染以及每次转染后的克隆选择。在此，我们描述了该系统的一种改进，即将tetP元件置于基于EBNA的附加体质粒中，该质粒可在灵长类细胞中稳定维持，但不能在啮齿类细胞中稳定维持。使用HeLa细胞和人黑色素瘤细胞，我们表明，瞬时或稳定转染后，报告基因以类似于原始系统的Tc调控方式表达。因此，该表达系统结合了附加体质粒载体的优点，如转染效率高和大量培养的时间高效选择，以及Tc调控系统提供的对基因表达的严格控制。
