Bäckman Cristina M, Zhang YaJun, Hoffer Barry J, Tomac Andreas C
Cellular Neurobiology Branch, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA.
J Neurosci Methods. 2004 Oct 30;139(2):257-62. doi: 10.1016/j.jneumeth.2004.05.012.
The most often used tetracycline-regulated transgenic mice system requires the generation of two transgenic strains, one carrying an inducible promoter and the other a transactivator. In this study, we report the design of a universal and simplified regulatory gene delivery vector to facilitate the generation of conditional transgenic animals that integrate both the tetracycline regulatory and response elements in a single vector. The newly developed tetracycline reversed transactivator rtTA-M2 was used in all our constructs, based on its highly improved properties with respect to specificity, stability and inducibility. To minimize interference between the different tetracycline-inducible promoters used in this study (tetracycline-responsive element (TRE), TRE-tight, or Tk-tetO) and the rtTA-M2 transactivator, both elements were cloned in opposite directions and separated by a 5 kb human p53 intron. The functionality of this system was confirmed after in vitro transfection in a mammalian cell line. Overall induction by the tetracycline-responsive element promoter was significantly higher than that induced by the newly developed TRE-tight promoter. However, the TRE-tight promoter showed a significantly tighter expression with minimal background, and still maintained high induction levels. The minimal Tk-tetO promoter showed a very weak induction capacity. Our study demonstrates that this combination of elements, placed in a single vector is sufficient for delivering a functional tetracycline-inducible system to a mammalian cell line. Moreover, additional modifications to this regulatory gene delivery system, such as the introduction of specific cloning sites and selection markers, have been designed with the idea of creating a simplified and universal inducible system to facilitate the generation of conditional transgenic, knock-out, and knock-in animals.
最常用的四环素调控转基因小鼠系统需要构建两种转基因品系，一种携带诱导型启动子，另一种携带反式激活因子。在本研究中，我们报告了一种通用且简化的调控基因递送载体的设计，以促进条件性转基因动物的产生，该载体将四环素调控元件和反应元件整合在一个单一载体中。我们在所有构建体中都使用了新开发的四环素反向反式激活因子rtTA-M2，因为它在特异性、稳定性和诱导性方面有了很大改进。为了尽量减少本研究中使用的不同四环素诱导型启动子（四环素反应元件（TRE）、TRE-紧密型或Tk-tetO）与rtTA-M2反式激活因子之间的干扰，这两个元件以相反方向克隆，并由一个5 kb的人p53内含子隔开。该系统的功能在哺乳动物细胞系的体外转染后得到了证实。四环素反应元件启动子的总体诱导明显高于新开发的TRE-紧密型启动子诱导的水平。然而，TRE-紧密型启动子显示出明显更严格的表达，背景最小，并且仍保持高诱导水平。最小的Tk-tetO启动子显示出非常弱的诱导能力。我们的研究表明，将这些元件组合置于一个单一载体中足以将一个功能性四环素诱导系统递送至哺乳动物细胞系。此外，对该调控基因递送系统进行了额外的修饰，如引入特定的克隆位点和选择标记，目的是创建一个简化且通用的诱导系统，以促进条件性转基因、敲除和敲入动物的产生。
