Molecular Biology and Genetics
|Electronic Journal of Biotechnology ISSN: 0717-3458
14 No. 2, Issue of March 15, 2011
|© 2011 by Pontificia Universidad Católica
de Valparaíso -- Chile
||Received October 27, 2010
/ Accepted January 19, 2011
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 ·
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: firstname.lastname@example.org
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.
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.