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Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 14, No. 2, 2011
Bioline Code: ej11023
Full paper language: English
Document type: Note
Document available free of charge

Electronic Journal of Biotechnology, Vol. 14, No. 2, 2011

 en Construction and application of a built-in dual luciferase reporter for microRNA functional analysis
Bi, Yanzhen; Zheng, Xinmin; Shao, Changwei; Pan, Wen; Jiang, Li & Ouyang, Huiwu

Abstract

Background: As key gene regulators, microRNAs post-transcriptionally modulate gene expression via binding to partially complementary sequence in the 3’ UTR of target mRNA. An accurate, rapid and quantitative tool for sensing and validation of miRNA targets is of crucial significance to decipher the functional implications of miRNAs in cellular pathways.
Results: Taking advantage of an improved restriction-free cloning method, we engineered a novel built-in dual luciferase reporter plasmid where Firefly and Renilla luciferase genes were assembled in a single plasmid named “pFila”. This design eliminates the transfection of a separate control plasmid and thus minimizes the time and labor required for miRNA-target sensing assays. pFila consistently produces Firefly and Renilla luciferase activities when transfected into human-, monkey- and mouse-derived mammalian cell systems. Moreover, pFila is capable of recapitulating the interaction of miR-16 and its known target CCNE1 in Hela cells. Additionally, pFila is shown to be a sensitive miR-biosensor by evaluating the inhibition efficiency of endogenous miRNA.
Conclusions: pFila would facilitate miRNA target identification and verification in a rapid and simplified manner. Also, pFila is a sensitive biosensor for active miRNA profiling in vivo.

Keywords
biosensor, luciferase, ligase-independent, miRNA, target.

 
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