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

文献类型： 外文期刊

作者： Bi, Yanzhen 1 ; Zheng, Xinmin 1 ; Shao, Changwei 2 ; Pan, Wen 3 ; Jiang, Li 2 ; Ouyang, Huiwu 2 ;

作者机构： 1.Hubei Acad Agr Sci, Inst Anim Husb & Vet, Hubei Key Lab Anim Embryo Engn & Mol Breeding, Wuhan, Peoples R China

2.Wuhan Univ, Coll Life Sci, RNA Grp, Wuhan, Peoples R China

3.Sun Yat Sen Univ, Affiliated Hosp 3, Guangzhou, Guangdong, Peoples R China

关键词： biosensor;luciferase;ligase-independent;miRNA;target

期刊名称：ELECTRONIC JOURNAL OF BIOTECHNOLOGY （ 影响因子：2.8； 五年影响因子：3.379 ）

ISSN： 0717-3458

年卷期： 2011 年 14 卷 2 期

页码：

收录情况： SCI

摘要： 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.

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