Fluorescent protein-based detection of φC31 integrase activity in mammalian cells

The enzyme φC31 integrase from Streptomyces phage has been documented as functional in mammalian cells and, therefore, has the potential to be a powerful gene manipulation tool. However, the activity of this enzyme is cell-type dependent. The more active mutant forms of φC31 integrase are required....

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Bibliographic Details
Published in:Analytical biochemistry 2013-10, Vol.441 (2), p.104-108
Main Authors: Liu, Taian, Fang, Yongxiang, Jia, Huaijie, Chen, Guohua, Guan, Qisai, He, Xiaobing, Yao, Wenjuan, Zeng, Shuang, Jing, Zhizhong
Format: Article
Language:English
Subjects:
Animals
bacteriophages
Bacteriophages - enzymology
Cell Line
Detecting method
enzyme activity
Enzyme Assays
enzymes
fluorescence
Fluorescent Dyes - analysis
Fluorescent Dyes - metabolism
Fluorescent proteins
Gene Expression
genes
green fluorescent protein
Green Fluorescent Proteins - analysis
Green Fluorescent Proteins - genetics
Humans
Integrase-inversion cassette
Integrases - genetics
Integrases - metabolism
Intracellular activity
Luminescent Proteins - analysis
Luminescent Proteins - genetics
mammals
mutants
protein synthesis
Streptomyces
Streptomyces - virology
Transfection
φC31 integrase
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Summary:The enzyme φC31 integrase from Streptomyces phage has been documented as functional in mammalian cells and, therefore, has the potential to be a powerful gene manipulation tool. However, the activity of this enzyme is cell-type dependent. The more active mutant forms of φC31 integrase are required. Therefore, a rapid and effective method should be developed to detect the intracellular activity of φC31 integrase. We devised in this study an integrase-inversion cassette that contains the enhanced green fluorescent protein (EGFP) gene and the reverse complementary DsRed gene, which are flanked by attB and reverse complementary attP. This cassette can be inverted by φC31 integrase, thereby altering the fluorescent protein expression. Thus, φC31 integrase activity can be qualitatively or quantitatively evaluated based on the detected fluorescence. Furthermore, this cassette-based method was applied to several cell types, demonstrating that it is an efficient and reliable tool for measuring φC31 integrase activity in mammalian cells.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2013.07.024