Enhanced snoMEN Vectors Facilitate Establishment of GFP-HIF-1[alpha] Protein Replacement Human Cell Lines

• Published in: PloS one 2016-04, Vol.11 (4)
• Main Authors: Ono, Motoharu, Yamada, Kayo, Bensaddek, Dalila, Afzal, Vackar, Biddlestone, John, Ortmann, Brian, Mudie, Sharon, Boivin, Vincent, Scott, Michelle S, Rocha, Sonia, Lamond, Angus I
• Format: Article
• Language: English
• Subjects: Analysis; Cellular proteins; Gene expression; Gene therapy; Genetic vectors; Small nuclear ribonucleoproteins
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0154759

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-1[alpha] 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.