Dual regulation of gene expression mediated by tetracycline and Cre recombinase.

BENCHMARKS Controlled gene expression is important for elucidating gene functions and developing safer methods of gene therapy (1,2). Historically, early inducible expression systems were mainly based on responses of endogenous elements to exogenous signals or stresses such as heat shock, hormones, or metal ions. However, these systems lacked specificity due to the inducer's pleiotropic effects. Consequently , nonmammalian or mutated endogenous control elements such as the lac repressor/operator, ecdysone-inducible system, Cre/Lox system, and tetracycline (Tc)-inducible system were used to achieve higher specificity of gene induction (1,2). Currently, the Tc-inducible system remains as one of the most commonly used expression systems (3). The Tc-inducible system is derived from regulatory Tc-resistant determinants in Gram-negative bacteria (4). There are 11 Tc resistance determinants , each consisting of a resistance protein (i.e., TetB) and a regulatory/ repressor protein (i.e., TetR). In the absence of Tc, TetR binds to the tet operator (tetO 2). However, the presence of Tc allows expression of Tc resistance proteins as TetR binds to Tc allosteri-cally and dissociates from tetO 2 (4). The best understood TetR protein is a repressor for the resistance protein TetB, or TetR(B) (or TetR henceforth) (5). TetR-regulated gene expression was first shown in plant cells (6) with subsequent advances that led to the development of Tc-responsive promoters for regulated gene expression in mam-malian cells (5,7). We have recently developed a single vector system, the so-called pTHE vector system, in which Tc-regulated gene expression is mediated by a chimeric repressor that recruits histone deacety-lases in mammalian cells (8). The chimeric repressor was engineered by fusing the TetR with an mSin3-interacting domain of human Mad1, which bound to tetO 2 element with high affinity and was abrogated by doxcycline, a commonly used stable analog of Tc. This Tc-inducible system improved the previous Tc-inducible systems by using a single vector that contained both the chimeric repressor and the Tc-responsive promoter (8). However, an apparent weakness of the pTHE system is that it requires time to produce the chimeric repressor, resulting in the lack of initial repression of the gene of interest. This could be highly problematic if a gene product is growth inhibitory or toxic. In order to improve the initial repression of transgene expression in our recently developed pTHE system (8), we inserted a LoxP-flanking stuffer containing the green fluorescent protein (GFP) coding sequence and a simian virus 40 (SV40) poly-adenylation (SV40 PA) signal into the pTHE …