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Archive > Volume 22 Issue 4 > 2015,22(4):420-426. DOI:10.3872/j.issn.1007-385X.2015.04.002 Prev Next
Prompt Report
The construction and application of an adenovirus mediated dual-luciferase reporter system
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Abstract:
Objective:To construct anadenovirus mediated dual-luciferase reportersystemthat can be used for screening promoters with high activity in a broad of cells. Methods: The testing promoter controlled expressioncassette of Firefly luciferase and CMV promoter controlled expressioncassette of Renilla luciferase were cloned into the E1 and E3 region of adenoviral vector respectively. The capacity of the system was assessed in the following three aspects: the activities of each promoter detected at different MOI, the variance within the same group, and the availability of the system for examining the promoter activity in cell lines that were hard to be transfected. Finally, the activities of CAG, CASI and a new CCAU promoters were examined by the system in 9 cell lines to find the promoter with high and broad-spectrum activity.Results: The adenovirus mediated dual-luciferase reporters system was successfully constructed. The activities of Rluc and Fluc of individual promoter had no significant difference at different MOI conditions(P>0.05), and the variations in each test group were small. The system worked effectively in K562, Jurkat, and primary skin cells, which are hard to be transfected. We found that the CCAU promoter produced higher luciferase activity than the CAG and CASI promoters in 9 cell lines tested (P<0.05 and P<0.01 respectively). Conclusion: The adenovirus mediated dual-luciferase reporters is an easy-to-use and reliable assay system suitable for assessing promoter activity. The new designed promoter CCAU has broad-spectrum strongactivity and can be used in transgenic over-expression.
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Project Supported:
Project supported by the Major Science and Technology Special Project for “Significant New Drug Creation” (No.2013ZX09102-060), and the Shanghai Engineering Technology Research Center Project (No.12DZ2251600)
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