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Nucleic Acids Research 2005 33(17):e147; doi:10.1093/nar/gni145
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Published online 4 October 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange

Ee Tsin Wong1,2, John L Kolman2, Yao-Cheng Li2, Larry D Mesner3, Wolfgang Hillen4, Christian Berens4 and Geoffrey M Wahl2,*

1Division of Biological Sciences, University of California at San Diego La Jolla, CA, USA 2The Salk Institute for Biological Studies La Jolla, CA, USA 3Department of Biochemistry and Molecular Genetics, University of Virginia Charlottesville, Virginia, USA 4Department of Microbiology, University of Erlangen-Nuremberg Erlangen, Germany

*To whom correspondence should be addressed. Tel: +858 453 4100; Fax: +858 457 2762; Email: wahl{at}salk.edu

Received July 7, 2005. Revised August 10, 2005. Accepted September 13, 2005.

Comparative analysis of mutants using transfection is complicated by clones exhibiting variable levels of gene expression due to copy number differences and genomic position effects. Recombinase-mediated cassette exchange (RMCE) can overcome these problems by introducing the target gene into pre-determined chromosomal loci, but recombination between the available recombinase targeting sites can reduce the efficiency of targeted integration. We developed a new LoxP site (designated L3), which when used with the original LoxP site (designated L2), allows highly efficient and directional replacement of chromosomal DNA with incoming DNA. A total of six independent LoxP integration sites introduced either by homologous recombination or retroviral delivery were analyzed; 70–80% of the clones analyzed in hamster and human cells were correct recombinants. We combined the RMCE strategy with a new, tightly regulated tetracycline induction system to produce a robust, highly reliable system for inducible transgene expression. We observed stable inducible expression for over 1 month, with uniform expression in the cell population and between clones derived from the same integration site. This system described should find significant applications for studies requiring high level and regulated transgene expression and for determining the effects of various stresses or oncogenic conditions in vivo and in vitro.


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