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Journal of Bacteriology, July 2002, p. 3657-3663, Vol. 184, No. 13
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.13.3657-3663.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Phage TP901-1 Site-Specific Integrase Functions in Human Cells

Stephanie M. Stoll, Daniel S. Ginsburg, and Michele P. Calos*

Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5120

Received 12 November 2001/ Accepted 31 March 2002

We demonstrate that the site-specific integrase encoded by phage TP901-1 of Lactococcus lactis subsp. cremoris has potential as a tool for engineering mammalian genomes. We constructed vectors that express this integrase in Escherichia coli and in mammalian cells and developed a simple plasmid assay to measure the frequency of intramolecular integration mediated by the integrase. We used the assay to document that the integrase functions efficiently in E. coli and determined that for complete reaction in E. coli, the minimal sizes of attB and attP are 31 and 50 bp, respectively. We carried out partial purification of TP901-1 integrase protein and demonstrated its functional activity in vitro in the absence of added cofactors, characterizing the time course and temperature optimum of the reaction. Finally, we showed that when expressed in human cells, the TP901-1 integrase carries out efficient intramolecular integration on a transfected plasmid substrate in the human cell environment. The TP901-1 phage integrase thus represents a new reagent for manipulating DNA in living mammalian cells.

* Corresponding author. Mailing address: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120. Phone: (650) 723-5558. Fax: (650) 723-5558. E-mail: calos{at}stanford.edu.

Journal of Bacteriology, July 2002, p. 3657-3663, Vol. 184, No. 13
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.13.3657-3663.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Ghosh, P., Bibb, L. A., Hatfull, G. F. (2008). Two-step site selection for serine-integrase-mediated excision: DNA-directed integrase conformation and central dinucleotide proofreading. Proc. Natl. Acad. Sci. USA 105: 3238-3243 [Abstract] [Full Text]  
  • Chen, L., Woo, S. L. C. (2005). Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cDNA. Proc. Natl. Acad. Sci. USA 102: 15581-15586 [Abstract] [Full Text]  
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