HUMAN GENE THERAPY 22:166–176 (February 2011)ª Mary Ann Liebert, Inc.DOI: 10.1089/hum.2010.099 Retroviral and Transposon-Based Tet-Regulated All-In-One Vectors with Reduced Background Expression and Improved Dynamic Range Niels Heinz,1 Axel Schambach,1 Melanie Galla,1 Tobias Maetzig,1 Christopher Baum,1 Rainer Loew,2 and Bernhard Schiedlmeier1 The regulated expression of therapeutic genes may become crucial in gene therapy when their constitutiveexpression interferes with cell fate in vivo. The efficient regulation of transgene expression requires tightly con-trolled inducible promoters, as shown for the tetracycline regulatory system (tet-system). However, its applicationrequires the introduction of two components into the target cell genome: the tet-responsive transactivator and theregulated expression cassette. In order to facilitate the usage of the tet-system for approaches in gene therapy, bothcomponents have to be transferred by a single vector, thus eliminating the preselection of transactivator positivecells. Published ‘‘all-in-one'' vectors for regulated transgene expression display a relatively low signal-to-noiseratio, resulting in regulatory windows of around 500-fold even in selected clones. In this study, we show that amodified vector architecture combined with the introduction of new tet-responsive promoters, Ptet, improved thedynamic range of such all-in-one vectors to levels up to 14,000-fold for viral and 25,000-fold for nonviral transfervectors in nonclonal human cell lines, and up to 2,800-fold in murine hematopoietic cell lines. This improvedregulation was the result of a strong reduction of background expression in the off-state, even if cells weretransduced at high multiplicity of infection, while induction remained at high levels. In addition, the resultsindicated that successful regulation of gene expression in different target cells depended on vector architecture aswell as the choice of the Ptet-promoter.