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

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...

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Autores principales:	Bi,Yanzhen, Zheng,Xinmin, Shao,Changwei, Pan,Wen, Jiang,Li, Ouyang,Huiwu
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2011
Materias:	biosensor luciferase ligase-independent miRNA target
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582011000200011
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spelling	oai:scielo:S0717-345820110002000112011-05-26Construction and application of a built-in dual luciferase reporter for microRNA functional analysisBi,YanzhenZheng,XinminShao,ChangweiPan,WenJiang,LiOuyang,Huiwu 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.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.14 n.2 20112011-03-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582011000200011en
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	biosensor luciferase ligase-independent miRNA target
spellingShingle	biosensor luciferase ligase-independent miRNA target Bi,Yanzhen Zheng,Xinmin Shao,Changwei Pan,Wen Jiang,Li Ouyang,Huiwu Construction and application of a built-in dual luciferase reporter for microRNA functional analysis
description	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.
author	Bi,Yanzhen Zheng,Xinmin Shao,Changwei Pan,Wen Jiang,Li Ouyang,Huiwu
author_facet	Bi,Yanzhen Zheng,Xinmin Shao,Changwei Pan,Wen Jiang,Li Ouyang,Huiwu
author_sort	Bi,Yanzhen
title	Construction and application of a built-in dual luciferase reporter for microRNA functional analysis
title_short	Construction and application of a built-in dual luciferase reporter for microRNA functional analysis
title_full	Construction and application of a built-in dual luciferase reporter for microRNA functional analysis
title_fullStr	Construction and application of a built-in dual luciferase reporter for microRNA functional analysis
title_full_unstemmed	Construction and application of a built-in dual luciferase reporter for microRNA functional analysis
title_sort	construction and application of a built-in dual luciferase reporter for microrna functional analysis
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2011
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582011000200011
work_keys_str_mv	AT biyanzhen constructionandapplicationofabuiltindualluciferasereporterformicrornafunctionalanalysis AT zhengxinmin constructionandapplicationofabuiltindualluciferasereporterformicrornafunctionalanalysis AT shaochangwei constructionandapplicationofabuiltindualluciferasereporterformicrornafunctionalanalysis AT panwen constructionandapplicationofabuiltindualluciferasereporterformicrornafunctionalanalysis AT jiangli constructionandapplicationofabuiltindualluciferasereporterformicrornafunctionalanalysis AT ouyanghuiwu constructionandapplicationofabuiltindualluciferasereporterformicrornafunctionalanalysis
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Construction and application of a built-in dual luciferase reporter for microRNA functional analysis

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