Phage R4 integrase mediates site-specific integration in human cells.
The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.
Olivares
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Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
Hollis
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RP
Calos
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D001287:Attachment Sites, Microbiological / Q000235:genetics*; D001435:Bacteriophages / Q000201:enzymology*; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D015894:Genome, Human; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D014162:Transfection; D016662:Virus Integration
D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D019426:Integrases; D009355:Neomycin