Molecular Biology and Genetics
  Animal Biotechnology
Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 14 No. 2, Issue of March 15, 2011
© 2011 by Pontificia Universidad Católica de Valparaíso -- Chile Received October 27, 2010 / Accepted January 19, 2011
DOI: 10.2225/vol14-issue2-fulltext-9  

Construction and application of a built-in dual luciferase reporter for microRNA functional analysis

Yanzhen Bi*1 · Xinmin Zheng1 · Changwei Shao2 · Wen Pan3 · Li Jiang2 · Huiwu Ouyang2

1Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Science, China
2RNA Group, College of Life Science, Wuhan University, China
3The Third Affiliated Hospital of Sun Yat-Sen University, Tianhe District, China

*Corresponding author:

Financial support: This work was funded by a research grant from Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding to Y. Z. Bi (2010ZD163).

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

Abstract   Full Text

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.

Supported by UNESCO / MIRCEN network