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Nucleic Acids Research Advance Access published online on May 22, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn298
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Regulated gene insertion by steroid-induced {Phi}C31 integrase

Nynne Sharma, Brian Moldt, Trine Dalsgaard, Thomas G. Jensen and Jacob Giehm Mikkelsen*

Department of Human Genetics, University of Aarhus, DK-8000 Aarhus C, Denmark

*To whom correspondence should be addressed. Tel: +45 89421651; Fax: +45 86123173; Email: giehm{at}humgen.au.dk

Received March 4, 2008. Revised April 4, 2008. Accepted April 29, 2008.

Nonviral integration systems are widely used genetic tools in transgenesis and play increasingly important roles in strategies for therapeutic gene transfer. Methods to efficiently regulate the activity of transposases and site-specific recombinases have important implications for their spatiotemporal regulation in live transgenic animals as well as for studies of their applicability as safe vectors for genetic therapy. In this report, strategies for posttranslational induction of a variety of gene-inserting proteins are investigated. An engineered hormone-binding domain, derived from the human progesterone receptor, hPR891, and specifically recognized by the synthetic steroid mifepristone, is fused to the Sleeping Beauty, Frog Prince, piggyBac and Tol2 transposases as well as to the Flp and {Phi}C31 recombinases. By analyzing mifepristone-directed inducibility of gene insertion in cultured human cells, efficient posttranslational regulation of the Flp recombinase and the {Phi}C31 integrase is documented. In addition, fusion of the {Phi}C31 integrase with the ERT2 modified estrogen receptor hormone-binding domain results in a protein, which is inducible by a factor of 22-fold and retains 75% of the activity of the wild-type protein. These inducible {Phi}C31 integrase systems are important new tools in transgenesis and in safety studies of the {Phi}C31 integrase for gene therapy applications.


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E. Sangiorgi, Z. Shuhua, and M. R. Capecchi
In vivo evaluation of PhiC31 recombinase activity using a self-excision cassette
Nucleic Acids Res., November 1, 2008; 36(20): e134 - e134.
[Abstract] [Full Text] [PDF]


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