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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

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Item Type:Article
Title:Enhanced efficiency through nuclear localization signal fusion on phage PhiC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells
Creators Name:Andreas, S. and Schwenk, F. and Kueter-Luks, B. and Faust, N. and Kuehn, R.
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.
Keywords:3T3 Cells, Bacteriophages, Cell Nucleus Active Transport, CHO Cells, Chromatin, DNA Nucleotidyltransferases, Genetic Engineering, Genetic Recombination, Integrases, Microbiological Attachment Sites, Nuclear Localization Signals, Recombinant Fusion Proteins, Streptomyces, Transfection, Viral Proteins, Animals, Cricetinae, Mice
Source:Nucleic Acids Research
ISSN:0305-1048
Publisher:Oxford University Press (U.K.)
Volume:30
Number:11
Page Range:2299-2306
Date:1 June 2002
Official Publication:https://doi.org/10.1093/nar/30.11.2299
PubMed:View item in PubMed

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