Liu H S, Lee C H, Lee C F, Su I J, Chang T Y
Department of Microbiology & Immunology, Medical College, National Cheng Kung University, Tainan, Taiwan, ROC.
Biotechniques. 1998 Apr;24(4):624-8, 630-2. doi: 10.2144/98244st03.
The Escherichia coli Lac repressor (Lac system) and tetracycline responsive promoter (Tet system) systems have been used individually to regulate gene expression at the cellular as well as the organismal levels. In this study, these two systems were combined (designated Lac/Tet dual-inducible system) to regulate two inducible genes simultaneously in a single cell. The isopropyl-beta-D-thiogalactopyranoside (IPTG) and tetracycline (used for the operation of the Lac and the Tet systems) were non-cytotoxic to the cells when added together into the cells at around the optimal concentrations (IPTG: < or = 5 mM; tetracycline: < 1.5 micrograms). The rate and efficiency of induction and repression of two inducible genes regulated by the Lac/Tet dual-inducible system were similar to the results obtained when one inducible gene is regulated by one inducible system in a single cell. The Lac/Tet dual-inducible system could function in many cell lines, which was demonstrated by regulating the expression of beta-galactosidase and luciferase reporter genes in five tumor cell lines by transient transfection analysis. The feasibility of introducing a second inducible system into an already established inducible cell line was confirmed. Finally, we showed that the Lac/Tet dual-inducible system functions at translational and at functional levels in a stable cell line named 7-4-b, which contains the Ha-ras and bc1-2 inducible genes. In conclusion, this study extends the application of prokaryotic inducible systems from the regulation of a single gene to two genes and helps clarify the relationship between two genes and the effects of two genes on the cells.
大肠杆菌乳糖阻遏物（乳糖系统）和四环素反应性启动子（四环素系统）已分别用于在细胞和生物体水平上调节基因表达。在本研究中，将这两个系统结合（命名为乳糖/四环素双诱导系统），以在单个细胞中同时调节两个诱导型基因。当以最佳浓度左右（异丙基-β-D-硫代半乳糖苷（IPTG）：≤5 mM；四环素：<1.5微克）一起添加到细胞中时，IPTG和四环素（用于乳糖和四环素系统的操作）对细胞无细胞毒性。由乳糖/四环素双诱导系统调节的两个诱导型基因的诱导和抑制速率及效率与单个细胞中一个诱导型系统调节一个诱导型基因时获得的结果相似。通过瞬时转染分析调节五种肿瘤细胞系中β-半乳糖苷酶和荧光素酶报告基因的表达，证明了乳糖/四环素双诱导系统可在许多细胞系中发挥作用。证实了将第二个诱导系统引入已建立的诱导细胞系的可行性。最后，我们表明乳糖/四环素双诱导系统在一个名为7-4-b的稳定细胞系中在翻译水平和功能水平上发挥作用，该细胞系含有Ha-ras和bc1-2诱导型基因。总之，本研究将原核诱导系统的应用从单个基因的调节扩展到两个基因的调节，并有助于阐明两个基因之间的关系以及两个基因对细胞的影响。
