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Enhanced efficiency through nuclear localization signal fusion on phage PhiC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells

Authors

  • S. Andreas
  • F. Schwenk
  • B. Kueter-Luks
  • N. Faust
  • R. Kuehn

Journal

  • Nucleic Acids Research

Citation

  • Nucleic Acids Res 30 (11): 2299-2306

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.


DOI

doi:10.1093/nar/30.11.2299