Department of Molecular Biology, Institute of Biological Sciences, Maria Curie-Skłodowska University, 20-033 Lublin, Poland.
Department of Biochemistry and Molecular Biology, Medical University of Lublin, 20-093 Lublin, Poland.
Int J Mol Sci. 2021 May 14;22(10):5206. doi: 10.3390/ijms22105206.
Precise analysis of the genetic expression and functioning of proteins requires experimental approaches that, among others, enable tight control of gene expression at the transcriptional level. Doxycycline-induced Tet-On/Tet-Off expression systems provide such an opportunity, and are frequently used to regulate the activity of genes in eukaryotic cells. Since its development, the Tet-system has evolved tight gene control in mammalian cells; however, some challenges are still unaddressed. In the current set up, the establishment of the standard Tet-based system in target cells is time-consuming and laborious and has been shown to be inefficient, especially in a long-term perspective. In this work, we present an optimized inducible expression system, which enables rapid generation of doxycycline-responsive cells according to a one- or two-step protocol. The reported modifications of the Tet-On system expand the toolbox for regulated mammalian gene expression and provide high, stable, and homogenous expression of the Tet-On3G transactivator, which is of fundamental importance in the regulation of transgenes.
精确分析蛋白质的基因表达和功能需要实验方法，这些方法能在转录水平上实现对基因表达的严格控制。强力霉素诱导的 Tet-On/Tet-Off 表达系统提供了这样的机会，并且常用于调节真核细胞中基因的活性。自其发展以来，Tet 系统已经在哺乳动物细胞中实现了严格的基因控制；然而，一些挑战仍未得到解决。在当前的设置中，在靶细胞中建立标准的 Tet 系统既耗时又费力，并且效率低下，尤其是从长期来看。在这项工作中，我们提出了一种优化的诱导表达系统，该系统可根据一步或两步方案快速生成强力霉素反应性细胞。所报道的 Tet-On 系统的修改扩展了受调控的哺乳动物基因表达的工具包，并提供了 Tet-On3G 转录激活物的高、稳定和同质表达，这对转基因的调控至关重要。
