Strathdee C A, McLeod M R, Hall J R
Gene Therapy and Molecular Virology Group, The John P. Robarts Research Institute, 100 Perth Drive, London, Ont., Canada.
Gene. 1999 Mar 18;229(1-2):21-9. doi: 10.1016/s0378-1119(99)00045-1.
The tetracycline-responsive expression system is based on the ability of the chimeric tTA and rtTA transactivators to stimulate specifically transcription from a companion synthetic CMV* or TK* promoter element, and can provide tightly regulated gene expression that can be induced up to five orders of magnitude in cultured cells and transgenic mice. A major problem with the system is that high level expression of the tTA or rtTA transactivators causes cellular toxicity. Under conditions of prolonged expression this results in selective pressure against the stable incorporation of vectors expressing the tTA or rtTA transactivators, and makes the generation of stable cell lines and transgenic mice problematic. In this report we describe the development of a set of autoregulated bi-directional expression vectors in which the weaker TK* promoter is used to direct expression of the rtTA or tTA transactivator and the stronger CMV* element is used to direct cDNA expression. In this format the transactivator and response elements are encoded on the same vector, which simplifies the system and ensures that gene expression is effectively skewed in favor of the cDNA while maintaining a continuously low level of transactivator expression. We find that such an autoregulated system works equally well for both the tTA and rtTA transactivators, provided that they contain a nuclear localization signal. Similar to other versions of the tetracycline-responsive expression system, gene expression is tightly regulated and can be efficiently switched between the off and on expression states by doxycycline. In contrast with other tetracycline-responsive systems, however, expression of the rtTA and tTA transactivators from the autoregulated TK* promoter is low enough such that there is no cellular toxicity associated with either expression state. By incorporating a selectable marker into these vectors, all of the components required for using the system are now contained on a single plasmid construct, and we find that this format provides a more reliable and greatly simplified method for the generation of stable cell lines.
四环素反应性表达系统基于嵌合反式四环素转录激活因子（tTA）和反向四环素转录激活因子（rtTA）反式激活剂特异性刺激与其配套的合成巨细胞病毒*（CMV*）或胸苷激酶*（TK*）启动子元件转录的能力，并且能够提供严格调控的基因表达，在培养细胞和转基因小鼠中其诱导水平可达五个数量级。该系统的一个主要问题是tTA或rtTA反式激活剂的高水平表达会导致细胞毒性。在长期表达的条件下，这会对表达tTA或rtTA反式激活剂的载体的稳定整合产生选择性压力，从而使稳定细胞系和转基因小鼠的构建出现问题。在本报告中，我们描述了一组自调控双向表达载体的构建，其中较弱的TK启动子用于指导rtTA或tTA反式激活剂的表达，而较强的CMV元件用于指导cDNA的表达。在这种形式下，反式激活剂和反应元件编码在同一载体上，这简化了系统，并确保基因表达有效地偏向于cDNA，同时保持反式激活剂的持续低水平表达。我们发现，只要tTA和rtTA反式激活剂含有核定位信号，这样的自调控系统对它们都同样有效。与四环素反应性表达系统的其他版本类似，基因表达受到严格调控，并且可以通过强力霉素在关闭和开启表达状态之间有效切换。然而，与其他四环素反应性系统不同的是，来自自调控TK*启动子的rtTA和tTA反式激活剂的表达足够低，以至于两种表达状态均无细胞毒性。通过将一个选择标记整合到这些载体中，使用该系统所需的所有组件现在都包含在单个质粒构建体中，并且我们发现这种形式为稳定细胞系的构建提供了一种更可靠且大大简化的方法。
