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Fluorescent protein-based detection of phi C31 integrase activity in mammalian cells

文献类型： 外文期刊

作者： Liu, Taian;Fang, Yongxiang;Jia, Huaijie;Chen, Guohua;Guan, Qisai;He, Xiaobing;Yao, Wenjuan;Zeng, Shuang;Jing, Zhizhong

作者机构：

关键词： Detecting method;phi C31 integrase;Intracellular activity;Integrase-inversion cassette;Fluorescent proteins

期刊名称： ANALYTICAL BIOCHEMISTRY

ISSN： 0003-2697

年卷期： 2013 年 441 卷 2 期

页码：

收录情况： SCI

摘要： The enzyme phi C31 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 phi C31 integrase are required. Therefore, a rapid and effective method should be developed to detect the intracellular activity of phi C31 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 phi C31 integrase, thereby altering the fluorescent protein expression. Thus, phi C31 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 phi C31 integrase activity in mammalian cells. (c) 2013 Elsevier Inc. All rights reserved.

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