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J Biotechnol. 2014 Jan;169:71-81. doi: 10.1016/j.jbiotec.2013.11.004. Epub 2013 Nov 16.

Viral vectors expressing a single microRNA-based short-hairpin RNA result in potent gene silencing in vitro and in vivo.

Author information

1
KU Leuven, Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, Leuven, Belgium.
2
KU Leuven, Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, Leuven, Belgium; KU Leuven, Leuven Viral Vector Core, Leuven, Belgium.
3
KU Leuven, Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, Leuven, Belgium; KU Leuven, Leuven Viral Vector Core, Leuven, Belgium.
4
KU Leuven, Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, Leuven, Belgium.
5
KU Leuven, Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, Leuven, Belgium. Electronic address: Veerle.Baekelandt@med.kuleuven.be.

Abstract

The characterization of RNA interference and the accompanying microRNAs (miRs), together with the exogenous expression of artificial miR-like elements, has led to the development of strategies for specific and potent gene silencing. In turn, this allows manipulation of gene expression levels for target validation purposes in cell culture or for the generation of animal models. In this study we determined the optimal strategy to achieve the most potent knockdown using miR-based viral vectors. We studied polycistronic miRs in a viral vector context and evaluated knockdown potency of multiple-miRs targeting the same seed sequence in parallel with miRs targeting different seed sequences, both for a reporter and endogenous mRNA targets. We demonstrate that potent knockdown can be obtained in vitro and in vivo using viral vectors that encode a single miR-based short-hairpin RNA and report a generic and effective cloning platform for artificial miR30-based short-hairpin RNAs to generate potent knockdown viral vectors.

KEYWORDS:

Adeno-associated viral vector; Dopaminergic neurons; Lentiviral vector; Multiple hairpins; RNA interference; miR30

PMID:
24252659
DOI:
10.1016/j.jbiotec.2013.11.004
[Indexed for MEDLINE]
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