seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 13, 2002: pp. 121–128 doi:10.1016/S1084–9521(02)00018-6, available online at http://www.idealibrary.com on Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling Zhou Zhu a,* , Tao Zheng a , Chun G. Lee a , Robert J. Homer b,1 and Jack A. Elias a Since the first tetracycline-controlled transcriptional activa- tion system was designed nearly a decade ago, new variants, modifications, and improvements have been steadily added to this powerful set of tools for temporal control of transgene expression in mammalian systems. Tetracycline-based externally regulatable (Tet-based) systems have been successfully used to control the expression of numerous transgenes in cultured cells and in whole organisms, especially in mice. The application of these systems has provided invaluable insights into the function and regulation of a variety of genes under physiological and pathological conditions. Because of the favorable characteristics of the inducing agent doxycycline and the efficiency and effectiveness of the operating mechanism, the Tet-based systems have attracted substantial attention from the transgenic research community and are rapidly gaining popularity. The original tetracycline-controlled transcriptional activator (tTA) is a regulator with tight control of target gene expression and a broad range of inducibility.Thereversetetracycline-controlledtranscriptional activator (rtTA) activates the responsive elements only in the presence of doxycycline, giving a convenient control over the target transgene. The recently developed tetracycline-controlled transcriptional silencer (tTS) has been successfully used in cultured cells and in transgenic mice. In combination with rtTA, tTS actively suppresses background expression or “leakiness” without impeding the inducibility of the target gene, providing a true “On/Off” transgenic switch. New variants of Tet-based regulators with improved features are still emerging and the utilities of these systems are constantly being tested. From the a Section of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520-8057,USAand b DepartmentofPathology,YaleUniversitySchoolof Medicine,NewHaven,CT06520-8057,USA. 1 Presentaddress:Pathology andLaboratoryMedicineService,VeteransAffairs,ConnecticutHealthCare System, West Haven, CT 06516, USA. * Corresponding author. E-mail: zhou.zhu@yale.edu © 2002 Elsevier Science Ltd. All rights reserved. 1084–9521 / 02 / $– see front matter Key words: transcriptional activation / gene regulation / transgene expression / temporal control / doxycycline © 2002 Elsevier Science Ltd. All rights reserved. Overview The completion of the human genome sequencing project is one of the most important achievements in science and human history. However, the func- tions of most of the estimated 40,000 protein-coding genes found in the human genome are virtually un- known.Thispresentstomolecularbiologists,biomed- ical scientists, and the entire research community a challenging task of elucidating the function and regulation of these genes under physiological and pathological circumstances. One powerful approach available to scientists is transgenic overexpression of genesofinterestinanimalmodelstostudytheroleof these genes in vivo. In the early 1980s, Gordon et al. first demonstrated that foreign DNA can be intro- duced into fertilized oocytes from a pregnant female mousebymicroinjection,andtheinjectedoocytescan be implanted into a pseudo-pregnant foster female mouse to generate progeny that carry a functional transgene. 1 Overthepasttwodecades,microinjection of fertilized eggs with DNA constructs has become the standard technology for generating transgenic animals. This has been successfully used to introduce numerous transgenes of interest into mice to gen- erate valuable information in understanding gene function. This approach has become widely available and easily accessible to scientists who are interested in establishing animal models for studying human diseases. The usefulness of tissue- or cell-specific promoters to directly control the expression of tar- get genes in the specified tissues in transgenic an- imals has been recognized and practiced wherever 121