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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Public Library of Science (PLoS)
2016-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0154759&type=printable |
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| author | Motoharu Ono Kayo Yamada Dalila Bensaddek Vackar Afzal John Biddlestone Brian Ortmann Sharon Mudie Vincent Boivin Michelle S Scott Sonia Rocha Angus I Lamond |
| author_facet | Motoharu Ono Kayo Yamada Dalila Bensaddek Vackar Afzal John Biddlestone Brian Ortmann Sharon Mudie Vincent Boivin Michelle S Scott Sonia Rocha Angus I Lamond |
| author_sort | Motoharu Ono |
| collection | DOAJ |
| 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. |
| format | Article |
| id | doaj-art-3f3efeaac1594ece912c3dd27558baf4 |
| institution | Kabale University |
| issn | 1932-6203 |
| language | English |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-3f3efeaac1594ece912c3dd27558baf42025-08-20T03:26:10ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01114e015475910.1371/journal.pone.0154759Enhanced snoMEN Vectors Facilitate Establishment of GFP-HIF-1α Protein Replacement Human Cell Lines.Motoharu OnoKayo YamadaDalila BensaddekVackar AfzalJohn BiddlestoneBrian OrtmannSharon MudieVincent BoivinMichelle S ScottSonia RochaAngus I LamondThe 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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0154759&type=printable |
| spellingShingle | Motoharu Ono Kayo Yamada Dalila Bensaddek Vackar Afzal John Biddlestone Brian Ortmann Sharon Mudie Vincent Boivin Michelle S Scott Sonia Rocha Angus I Lamond Enhanced snoMEN Vectors Facilitate Establishment of GFP-HIF-1α Protein Replacement Human Cell Lines. PLoS ONE |
| 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 |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0154759&type=printable |
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