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Coordinate lentiviral expression of Cre recombinase and RFP/EGFP mediated by FMDV 2A and analysis of Cre activity
Author(s) -
Liu Kuancan,
Wang Heng,
Long Ying,
Ye Jun,
Yuan Li
Publication year - 2012
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.24168
Subject(s) - cre recombinase , green fluorescent protein , viral vector , biology , vector (molecular biology) , reporter gene , microbiology and biotechnology , expression cassette , transgene , recombinase , recombinant dna , gene expression , gene , genetics , genetically modified mouse , recombination
Abstract The site‐specific recombination mediated by Cre recombinase has been utilized extensively in genetic engineering and gene function studies. Efficient delivery of a Cre enzyme with enzymatic activity and the ability to monitor the enzyme expression are required in applications, and lentiviral constructs with a fluorescent protein (FP) to report the Cre expression are suitable for most studies. However, the current lentiviral vector systems have some deficiencies in precise reporting the Cre expression through fluorescence. To solve the problem, we generated a lentiviral system with Cre and RFP or EGFP bridged by an FMDV 2A sequence in an open reading frame expressed by a CMV promoter. We then examined the capabilities of the constructs to package with VSVG into infectious virus and to mediate expression of the Cre enzyme and fluorescent reporter. Furthermore, we monitored the bioactivities of the expressed products. We demonstrated the coordinate expression of the enzyme and the reporter. The expressed Cre was efficient at removing LoxP‐flanked fragments in cells and did not show obvious cellular toxicity, and the expressed FPs allowed direct observation under fluorescent microscope. Therefore, the conjugation of CMV‐Cre‐2A‐FP represents a significant improvement to the current lentiviral Cre delivery systems for obtaining a required Cre activity while accurately monitoring its presence. Our study also provides information concerning application of the established vector system. J. Cell. Biochem. 113: 2909–2919, 2012. © 2012 Wiley Periodicals, Inc.