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Published online on May 9, 2000, 10.1073/pnas.090527097
PNAS | May 23, 2000 | vol. 97 | no. 11 | 5995-6000


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Genetics
 A phage integrase directs efficient site-specific integration in human cells

Amy C. Groth*, Eric C. Olivares*, Bhaskar Thyagarajan, and Michele P. Calosdagger

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

Edited by Allan Campbell, Stanford University, Stanford, CA, and approved March 2, 2000 (received for review December 3, 1999)

The integrase from the Streptomyces phage phi C31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the phi C31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein-Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.

* A.C.G. and E.C.O. contributed equally to this work.

dagger To whom reprint requests should be addressed. E-mail: calos{at}stanford.edu.


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