REGULATED EXPRESSION OF FOREIGN GENES IN MAMMALIAN-CELLS UNDER THE CONTROL OF COLIPHAGE-T3 RNA-POLYMERASE AND LAC REPRESSOR
REGULATED EXPRESSION OF FOREIGN GENES IN MAMMALIAN-CELLS UNDER THE CONTROL OF COLIPHAGE-T3 RNA-POLYMERASE AND LAC REPRESSOR
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DOI：
10.1073/pnas.86.14.5400
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发表时间：
1989-07-01
影响因子：
11.1
通讯作者：
BUJARD, H
中科院分区：
文献类型：
--
作者：
DEUSCHLE, U;PEPPERKOK, R;BUJARD, H
在复杂的遗传背景下严格调控单个基因表达的系统极大地促进了基因功能的分析。在这份报告中，在哺乳动物细胞中的高度调节的表达系统的发展进行了描述，其中外源基因的转录介导的噬菌体T3 RNA聚合酶的大肠杆菌乳糖阻遏物的控制下。已经建立了组成型表达噬菌体RNA聚合酶和乳糖阻遏物的兔肾细胞系。这两种细菌蛋白调节萤火虫荧光素酶的编码序列的转录，所述萤火虫荧光素酶已被置于T3启动子/lac操纵子融合体的控制下。在诱导剂异丙基β-的存在下观察到D-硫代半乳糖苷有效的T3聚合酶依赖性转录，其在不存在诱导剂的情况下被紧密抑制。T3转录物的翻译可由牛痘病毒功能介导。在高等真核细胞中，通过使用高度特异性和无毒的诱导剂，可以在几个数量级的范围内调节特定的转录活性，这一证明对各种研究具有广泛的意义。
Systems that stringently regulate the expression of individual genes within a complex genetic background have contributed greatly to the analysis of gene function. In this report the development of a highly regulated expression system in mammalian cells is described in which transcription of a foreign gene is mediated by the bacteriophage T3 RNA polymerase under the control of the Escherichia coli lac repressor. Rabbit kidney cell lines have been established that constitutively express the phage RNA polymerase and lac repressor. The two bacterial proteins regulate the transcription of the coding sequence of the firefly luciferase, which has been placed under the control of a T3 promoter/lac operator fusion. In the presence of the inducer isopropyl .beta.-D-thiogalactoside, efficent T3 polymerase-dependent transcription is observed, which is tightly repressed in the absence of inducer. Translation of the T3 transcripts can be mediated by vaccinia virus functions. The demonstration that a specific transcription activity can be regulated over a range of several orders of magnitude in higher eukaryotic cells by using a highly specific and nontoxic inducer has broad implications for a variety of studies.