Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Jiwaji, M. et al. (2014) Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells. PLoS ONE, 9(6), e99458. (doi:10.1371/journal.pone.0099458) (PMID:24926959) (PMCID:PMC4057226)

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Abstract

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Jiwaji, Dr Meesbah and Kolch, Prof Walter and Sandison, Dr Mairi and Cooper, Professor Jonathan and Reboud, Dr Julien and Pitt, Dr Andrew and Barkess, Dr Grainne and Girolami, Prof Mark and Daly, Dr Ronan
Authors: Jiwaji, M., Sandison, M. E., Reboud, J., Stevenson, R., Daly, R., Barkess, G., Faulds, K., Kolch, W., Graham, D., Girolami, M. A., Cooper, J. M., and Pitt, A. R.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
College of Science and Engineering > School of Computing Science
College of Science and Engineering > School of Engineering > Biomedical Engineering
Journal Name:PLoS ONE
Publisher:Public Library of Science
ISSN:1932-6203
ISSN (Online):1932-6203
Copyright Holders:Copyright © 2014 The Authors
First Published:First published in PLoS One 9(6):e99458
Publisher Policy:Reproduced under a Creative Commons License
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
436881The molecular noseAndrew PittEngineering & Physical Sciences Research Council (EPSRC)EP/E032745/1RI MOLECULAR CELL & SYSTEMS BIOLOGY