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

Motoharu Ono, Kayo Ono, Dalila Bensaddek, Vackar Afzal, John Biddlestone, Brian Ortmann, Sharon Mudie, Vincent Boivin, Michelle S. Scott, Sonia Rocha, Angus I. Lamond (Lead / Corresponding author)

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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.
Original languageEnglish
Article numbere0154759
Number of pages28
JournalPLoS ONE
Volume11
Issue number4
DOIs
Publication statusPublished - 29 Apr 2016

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    Ono, M., Ono, K., Bensaddek, D., Afzal, V., Biddlestone, J., Ortmann, B., Mudie, S., Boivin, V., S. Scott, M., Rocha, S., & Lamond, A. I. (2016). Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines. PLoS ONE, 11(4), [e0154759]. https://doi.org/10.1371/journal.pone.0154759