Enhanced snoMEN vectors facilitate establishment of GFP–HIF-1α protein replacement human cell lines

Granneman, S. et al. (2016) Enhanced snoMEN vectors facilitate establishment of GFP–HIF-1α protein replacement human cell lines. PLoS ONE, 11(4), e0154759. (doi: 10.1371/journal.pone.0154759) (PMID:27128805) (PMCID:PMC4851398)

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: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Biddlestone, Dr John
• Authors: Granneman, S., Ono, M., Yamada, K., Bensaddek, D., Afzal, V., Biddlestone, J., Ortmann, B., Mudie, S., Boivin, V., Scott, M. S., Rocha, S., and Lamond, A. I.
• College/School: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
• Journal Name: PLoS ONE
• Publisher: Public Library of Science
• ISSN: 1932-6203
• ISSN (Online): 1932-6203
• Copyright Holders: Copyright © 2016 Ono et al.
• First Published: First published in PLoS ONE 11(4):e0154759
• Publisher Policy: Reproduced under a Creative Commons License