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Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.

机译:四环素反应性启动子可严格控制哺乳动物细胞中的基因表达。

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摘要

Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such genes in a reversible way.
机译:在大肠杆菌的Tn10中编码的四环素抗性操纵子的控制元件已被用来在哺乳动物细胞中建立高效的调控系统。通过将tet阻遏物与单纯疱疹病毒的病毒体蛋白16的激活域融合,生成了在HeLa细胞中组成型表达的四环素控制的反式激活因子(tTA)。该反式激活因子可刺激源自人类巨细胞病毒启动子IE的最小启动子序列与tet操纵子序列的结合。将受tTA依赖性启动子控制的萤光素酶基因整合到产生tTA的HeLa细胞系中后,就可以监测高水平的萤光素酶表达。这些活动对四环素敏感。根据培养基中抗生素的浓度(0-1微克/毫升),萤光素酶的活性可以调节到五个数量级。因此,该系统不仅允许差异控制哺乳动物细胞中单个基因的活性,而且适用于以可逆方式为此类基因创建“开/关”情况。

著录项

  • 作者

    Gossen, M; Bujard, H;

  • 作者单位
  • 年度 1992
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  • 原文格式 PDF
  • 正文语种 en
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