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PLoS One. 2016 Apr 29;11(4):e0154759. doi: 10.1371/journal.pone.0154759. eCollection 2016.

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

Author information

1
Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
2
Department of Biochemistry and RNA Group, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Canada.

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.

PMID:
27128805
PMCID:
PMC4851398
DOI:
10.1371/journal.pone.0154759
[Indexed for MEDLINE]
Free PMC Article

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