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Nucleic Acids Research Advance Access originally published online on December 20, 2007
Nucleic Acids Research 2008 36(1):e9; doi:10.1093/nar/gkm1123
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Nucleic Acids Research, 2008, Vol. 36, No. 1 e9
© 2007 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.

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Site-specific recombination in Schizosaccharomyces pombe and systematic assembly of a 400kb transgene array in mammalian cells using the integrase of Streptomyces phage {phi}BT1

Zhengyao Xu, Nicholas C. O. Lee, Felix Dafhnis-Calas, Sunir Malla, Margaret C. M. Smith and William R. A. Brown*

Institute of Genetics, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK

*To whom correspondence should be addressed: Tel: +44 (0)115 823 0386; Fax: +44 (0)115 823 0338; Email: William.brown{at}nottingham.ac.uk

Received August 28, 2007. Revised November 10, 2007. Accepted December 3, 2007.

We have established the integrase of the Streptomyces phage {phi}BT1 as a tool for eukaryotic genome manipulation. We show that the {phi}BT1 integrase promotes efficient reciprocal and conservative site-specific recombination in vertebrate cells and in Schizosaccharomyces pombe, thus establishing the utility of this protein for genome manipulation in a wide range of eukaryotes. We show that the {phi}BT1 integrase can be used in conjunction with Cre recombinase to promote the iterative integration of transgenic DNA. We describe five cycles of iterative integration of a candidate mouse centromeric sequence 80 kb in length into a human mini-chromosome within a human-Chinese hamster hybrid cell line. These results establish the generality of the iterative site-specific integration technique.

Present address: Margaret C. M. Smith, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK


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