Improved applications of the tetracycline-regulated gene depletion system.
Improved applications of the tetracycline-regulated gene depletion system.
复制标题
DOI：
--
复制
发表时间：
2009-10
影响因子：
5.5
通讯作者：
H. Nishijima;T. Yasunari;Tatsuo Nakayama;N. Adachi;K. Shibahara
中科院分区：
文献类型：
--
作者：
H. Nishijima;T. Yasunari;Tatsuo Nakayama;N. Adachi;K. Shibahara
严格控制转基因在哺乳动物细胞中的表达是生物学研究、药物发现和未来基因治疗的重要工具。四环素调控的基因缺失（Tet-Off）系统已被广泛用于控制哺乳动物细胞中的基因活性，因为它允许对转基因进行严格调控，但不具有原核调节蛋白的多效性。然而，Tet-Off系统并不与每种细胞类型兼容，这是该系统剩下的主要障碍。最近，我们通过插入一个内部核糖体进入位点（IRES）来驱动来自同一个四环素应答启动子的转基因选择标记，从而克服了这个问题。我们还采用了CMV立即早期增强子/β-肌动蛋白（CAG）启动子来表达Tet-controlled transactivator。事实上，经过这些技术修改的Tet-Off系统已成功应用于人类pre-B Nalm-6细胞系，而传统的Tet-Off系统在该细胞系中无法有效工作。这些方法的改进应该适用于许多其他哺乳动物的增殖细胞。在这篇综述中，我们给出了一个概述，并介绍了一种新的方法，用于改进应用的Tet-Off系统。
Tightly controlled expression of transgenes in mammalian cells is an important tool for biological research, drug discovery, and future genetic therapies. The tetracycline-regulated gene depletion (Tet-Off) system has been widely used to control gene activities in mammalian cells, because it allows strict regulation of transgenes but no pleiotropic effects of prokaryotic regulatory proteins. However, the Tet-Off system is not compatible with every cell type and this is the main remaining obstacle left for this system. Recently, we overcame this problem by inserting an internal ribosome entry site (IRES) to drive a selectable marker from the same tetracycline-responsive promoter for the transgene. We also employed a CMV immediate early enhancer/beta-actin (CAG) promoter to express a Tet-controlled transactivator. Indeed, the Tet-Off system with these technical modifications was applied successfully to the human pre-B Nalm-6 cell line in which conventional Tet-Off systems had not worked efficiently. These methodological improvements should be applicable for many other mammalian proliferating cells. In this review we give an overview and introduce a new method for the improved application of the Tet-Off system.