title: Enhanced snoMEN Vectors Facilitate Establishment of GFP-HIF-1α Protein Replacement Human Cell Lines.
abstract: 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.
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Motoharu
authors.initials: M
authors.lastname: Ono
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Kayo
authors.initials: K
authors.lastname: Yamada
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Dalila
authors.initials: D
authors.lastname: Bensaddek
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Vackar
authors.initials: V
authors.lastname: Afzal
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: Biddlestone
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Brian
authors.initials: B
authors.lastname: Ortmann
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Sharon
authors.initials: S
authors.lastname: Mudie
authors.affiliation: Department of Biochemistry and RNA Group, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Canada.
authors.forename: Vincent
authors.initials: V
authors.lastname: Boivin
authors.affiliation: Department of Biochemistry and RNA Group, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Canada.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Scott
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Sonia
authors.initials: S
authors.lastname: Rocha
authors.affiliation: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Angus I
authors.initials: AI
authors.lastname: Lamond
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020537:RNA, Small Nucleolar; D011993:Recombinant Fusion Proteins; D020718:Ribonucleoproteins, Small Nucleolar; D049452:Green Fluorescent Proteins