BioScience Trends. 2009;3(5):161-167. (DOI: )

Improved applications of the tetracycline-regulated gene depletion system.

Nishijima H, Yasunari T, Nakayama T, Adachi N, Shibahara K

Tightly controlled expression of transgenes in mammalian cells is an important tool for biological research, drug discovery, and future genetic therapies. The tetracyclineregulated 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/β-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.

Full Text: