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

Abstract

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.

Keywords: Detecting method; Fluorescent proteins; Integrase-inversion cassette; Intracellular activity; φC31 integrase.