An improved vector system for homogeneous and stable gene regulation

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.