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 ORCID: 0000-0002-3639-0691, Mudie, Sharon, Boivin, Vincent ORCID: 0000-0001-6434-2737, Scott, Michelle S, Rocha, Sonia ORCID: 0000-0002-2413-4981 et al (show 1 more authors) (2016) Enhanced snoMEN Vectors Facilitate Establishment of GFP-HIF-1α Protein Replacement Human Cell Lines. PloS one, 11 (4). e0154759-.

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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.

Item Type: Article
Uncontrolled Keywords: Cell Line, Hela Cells, Humans, Ribonucleoproteins, Small Nucleolar, Green Fluorescent Proteins, Recombinant Fusion Proteins, RNA, Small Nucleolar, Gene Expression, Base Sequence, Nucleic Acid Conformation, Protein Binding, Genetic Vectors, Hypoxia-Inducible Factor 1, alpha Subunit, Gene Knockdown Techniques
Depositing User: Symplectic Admin
Date Deposited: 10 Jan 2018 10:13
Last Modified: 19 Jan 2023 06:46
DOI: 10.1371/journal.pone.0154759
Open Access URL: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC48513...
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URI: https://livrepository.liverpool.ac.uk/id/eprint/3015773
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