Cargando…

Polymerase II Promoter Strength Determines Efficacy of microRNA Adapted shRNAs

Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymer...

Descripción completa

Detalles Bibliográficos
Autores principales: Lebbink, Robert Jan, Lowe, Maggie, Chan, Theresa, Khine, Htet, Wang, Xiaoyin, McManus, Michael T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198731/
https://www.ncbi.nlm.nih.gov/pubmed/22031824
http://dx.doi.org/10.1371/journal.pone.0026213
_version_ 1782214481750786048
author Lebbink, Robert Jan
Lowe, Maggie
Chan, Theresa
Khine, Htet
Wang, Xiaoyin
McManus, Michael T.
author_facet Lebbink, Robert Jan
Lowe, Maggie
Chan, Theresa
Khine, Htet
Wang, Xiaoyin
McManus, Michael T.
author_sort Lebbink, Robert Jan
collection PubMed
description Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs). Although shRNA-miRs are effective in directing target mRNA knockdown and hence reducing protein expression in many cell types, variability of RNAi efficacy in cell lines has been an issue. Here we show that the choice of the polymerase II promoter used to drive shRNA expression is of critical importance to allow effective mRNA target knockdown. We tested the abundance of shRNA-miRs expressed from five different polymerase II promoters in 6 human cell lines and measured their ability to drive target knockdown. We observed a clear positive correlation between promoter strength, siRNA expression levels, and protein target knockdown. Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells. Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.
format Online
Article
Text
id pubmed-3198731
institution National Center for Biotechnology Information
language English
publishDate 2011
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-31987312011-10-26 Polymerase II Promoter Strength Determines Efficacy of microRNA Adapted shRNAs Lebbink, Robert Jan Lowe, Maggie Chan, Theresa Khine, Htet Wang, Xiaoyin McManus, Michael T. PLoS One Research Article Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs). Although shRNA-miRs are effective in directing target mRNA knockdown and hence reducing protein expression in many cell types, variability of RNAi efficacy in cell lines has been an issue. Here we show that the choice of the polymerase II promoter used to drive shRNA expression is of critical importance to allow effective mRNA target knockdown. We tested the abundance of shRNA-miRs expressed from five different polymerase II promoters in 6 human cell lines and measured their ability to drive target knockdown. We observed a clear positive correlation between promoter strength, siRNA expression levels, and protein target knockdown. Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells. Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type. Public Library of Science 2011-10-21 /pmc/articles/PMC3198731/ /pubmed/22031824 http://dx.doi.org/10.1371/journal.pone.0026213 Text en Lebbink et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Lebbink, Robert Jan
Lowe, Maggie
Chan, Theresa
Khine, Htet
Wang, Xiaoyin
McManus, Michael T.
Polymerase II Promoter Strength Determines Efficacy of microRNA Adapted shRNAs
title Polymerase II Promoter Strength Determines Efficacy of microRNA Adapted shRNAs
title_full Polymerase II Promoter Strength Determines Efficacy of microRNA Adapted shRNAs
title_fullStr Polymerase II Promoter Strength Determines Efficacy of microRNA Adapted shRNAs
title_full_unstemmed Polymerase II Promoter Strength Determines Efficacy of microRNA Adapted shRNAs
title_short Polymerase II Promoter Strength Determines Efficacy of microRNA Adapted shRNAs
title_sort polymerase ii promoter strength determines efficacy of microrna adapted shrnas
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198731/
https://www.ncbi.nlm.nih.gov/pubmed/22031824
http://dx.doi.org/10.1371/journal.pone.0026213
work_keys_str_mv AT lebbinkrobertjan polymeraseiipromoterstrengthdeterminesefficacyofmicrornaadaptedshrnas
AT lowemaggie polymeraseiipromoterstrengthdeterminesefficacyofmicrornaadaptedshrnas
AT chantheresa polymeraseiipromoterstrengthdeterminesefficacyofmicrornaadaptedshrnas
AT khinehtet polymeraseiipromoterstrengthdeterminesefficacyofmicrornaadaptedshrnas
AT wangxiaoyin polymeraseiipromoterstrengthdeterminesefficacyofmicrornaadaptedshrnas
AT mcmanusmichaelt polymeraseiipromoterstrengthdeterminesefficacyofmicrornaadaptedshrnas