Construction and application of a built-in dual luciferase reporter for microRNA functional analysis
Document title: Construction and application of a built-in dual luciferase reporter for microRNA functional analysis
Journal: Electronic journal of biotechnology
Database: PERIÓDICA
System number: 000336255
ISSN: 0717-3458
Authors: 1
1
2
3
2
2
Institutions: 1Hubei Academy of Agricultural Science, Institute of Animal Husbandry and Veterinary, Wuhan, Hubei. China
2Wuhan University, College of Life Science, Wuhan, Hubei. China
3Sun Yat-Sen University, The Third Affiliated Hospital, Tianhe. China
Year:
Season: Mar
Volumen: 14
Number: 2
Country: Chile
Language: Inglés
Document type: Artículo
Approach: Experimental, aplicado
English 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
Disciplines: Biología
Keyword: Genética,
Biotecnología,
Expresión génica,
Transcripción genética,
ARN mitocondrial,
Biosensores,
Luciferasa
Keyword: Biology,
Genetics,
Biotechnology,
Gene expression,
Genetic transcription,
Mitochondrial RNA,
Biosensors,
Luciferase
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