Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Generation of Cell Lines with Tetracycline-Regulated Gene Expression

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The expression of transfected genes can be regulated by systems that are sensitive to tetracycline (see the chapter introduction for details). The following protocol is based on Clontech's Tet-On1 3G line of tetracycline-inducible expression systems. These systems incorporate a Tet-On 3G transactivator protein consisting of an rtTA that has been optimized for effector sensitivity using an enforced retroviral evolution strategy in human cells. The components of the Tet-On system were incorporated into the human immunodeficiency virus (HIV)-1 genome so that expression of viral proteins and hence viral replication was dependent on doxycycline (Dox) administration (Zhou et al. 2006). Prolonged culturing of the HIV-rtTA virus resulted in virus variants that acquired mutations in the rtTA protein, enhancing transcriptional activity by up to sevenfold and Dox sensitivity by nearly 100-fold. The Tet-responsive promoter (here referred to as PTRE3G) consists of the minimal CMV promoter altered by the systematic introduction of a series of rational modifications to further minimize background expression (Loew et al. 2010). The Tet-On 3G transactivator protein binds to and activates genes preceded by the PTRE3G at very low concentrations of Dox (510 ng/mL) and can yield up to a 27,000-fold difference in expression between induced and uninduced cells in certain cell lines (see http://www.clontech.com; Resource Application Notes: Tet-On 3G Inducible Expression SystemLowest Background, Highest Sensitivity).


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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