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Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human...

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Autores principales: Ono, Motoharu, Yamada, Kayo, Bensaddek, Dalila, Afzal, Vackar, Biddlestone, John, Ortmann, Brian, Mudie, Sharon, Boivin, Vincent, Scott, Michelle S., Rocha, Sonia, Lamond, Angus I.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851398/
https://www.ncbi.nlm.nih.gov/pubmed/27128805
http://dx.doi.org/10.1371/journal.pone.0154759
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author Ono, Motoharu
Yamada, Kayo
Bensaddek, Dalila
Afzal, Vackar
Biddlestone, John
Ortmann, Brian
Mudie, Sharon
Boivin, Vincent
Scott, Michelle S.
Rocha, Sonia
Lamond, Angus I.
author_facet Ono, Motoharu
Yamada, Kayo
Bensaddek, Dalila
Afzal, Vackar
Biddlestone, John
Ortmann, Brian
Mudie, Sharon
Boivin, Vincent
Scott, Michelle S.
Rocha, Sonia
Lamond, Angus I.
author_sort Ono, Motoharu
collection PubMed
description The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins. Using multiplex 47snoMEM vectors that co-express multiple 47snoMEN in a single transcript, each of which can target different sites in the same mRNA, we document >3-fold increase in knock-down efficiency when compared with the original HBII-180C based snoMEN. The multiplex 47snoMEM vector allowed the construction of human protein replacement cell lines with improved efficiency, including the establishment of novel GFP–HIF-1α replacement cells. Quantitative mass spectrometry analysis confirmed the enhanced efficiency and specificity of protein replacement using the 47snoMEN-PR vectors. The 47snoMEN vectors expand the potential applications for snoMEN technology in gene expression studies, target validation and gene therapy.
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spelling pubmed-48513982016-05-07 Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines Ono, Motoharu Yamada, Kayo Bensaddek, Dalila Afzal, Vackar Biddlestone, John Ortmann, Brian Mudie, Sharon Boivin, Vincent Scott, Michelle S. Rocha, Sonia Lamond, Angus I. PLoS One Research Article The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins. Using multiplex 47snoMEM vectors that co-express multiple 47snoMEN in a single transcript, each of which can target different sites in the same mRNA, we document >3-fold increase in knock-down efficiency when compared with the original HBII-180C based snoMEN. The multiplex 47snoMEM vector allowed the construction of human protein replacement cell lines with improved efficiency, including the establishment of novel GFP–HIF-1α replacement cells. Quantitative mass spectrometry analysis confirmed the enhanced efficiency and specificity of protein replacement using the 47snoMEN-PR vectors. The 47snoMEN vectors expand the potential applications for snoMEN technology in gene expression studies, target validation and gene therapy. Public Library of Science 2016-04-29 /pmc/articles/PMC4851398/ /pubmed/27128805 http://dx.doi.org/10.1371/journal.pone.0154759 Text en © 2016 Ono 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 (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Ono, Motoharu
Yamada, Kayo
Bensaddek, Dalila
Afzal, Vackar
Biddlestone, John
Ortmann, Brian
Mudie, Sharon
Boivin, Vincent
Scott, Michelle S.
Rocha, Sonia
Lamond, Angus I.
Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines
title Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines
title_full Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines
title_fullStr Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines
title_full_unstemmed Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines
title_short Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines
title_sort enhanced snomen vectors facilitate establishment of gfp–hif-1α protein replacement human cell lines
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851398/
https://www.ncbi.nlm.nih.gov/pubmed/27128805
http://dx.doi.org/10.1371/journal.pone.0154759
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