title: Enhanced efficiency through nuclear localization signal fusion on phage PhiC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells.
abstract: The integrase of the phage PhiC31 recombines an attP site in the phage genome with a chromosomal attB site of its Streptomyces host. We have utilized the integrase-mediated reaction to achieve episomal and genomic deletion of a reporter gene in mammalian cells, and provide the first comparison of its efficiency with other recombinases in a new assay system. This assay demonstrated that the efficiency of PhiC31-integrase is significantly enhanced by the C-terminal, but not the N-terminal, addition of a nuclear localization signal and becomes comparable with that of the widely used Cre/loxP system. Furthermore, we found that the improved FLP recombinase, FLPe, exhibits only 10% recombination activity on chromosomal targets as compared with Cre, whereas the Anabaena derived XisA recombinase is essentially inactive in mammalian cells. These results provide the first demonstration that a nuclear localisation signal and its position within a recombinase can be important for its efficiency in mammalian cells and establish the improved PhiC31-integrase as a new tool for genome engineering.
authors.affiliation: Artemis Pharmaceuticals GmbH, Neurather Ring 1, 51063 Köln, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Andreas
authors.forename: Frieder
authors.initials: F
authors.lastname: Schwenk
authors.forename: Birgit
authors.initials: B
authors.lastname: Küter-Luks
authors.forename: Nicole
authors.initials: N
authors.lastname: Faust
authors.forename: Ralf
authors.initials: R
authors.lastname: Kühn
mesh_terms: D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus; D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D001435:Bacteriophages / Q000201:enzymology*; D016466:CHO Cells; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D005818:Genetic Engineering; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D019913:Nuclear Localization Signals / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic; D013302:Streptomyces / Q000821:virology; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002843:Chromatin; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases