KU Leuven, Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, Leuven, Belgium.
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.
J Biotechnol. 2014 Jan;169:71-81. doi: 10.1016/j.jbiotec.2013.11.004. Epub 2013 Nov 16.
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.
RNA 干扰的特性和伴随的 microRNAs（miRs），以及人工 miR 样元件的外源表达，已经导致了特定和有效的基因沉默策略的发展。反过来，这允许为细胞培养中的靶标验证目的或为动物模型的产生来操纵基因表达水平。在这项研究中，我们确定了使用基于 miR 的病毒载体实现最有效基因敲低的最佳策略。我们在病毒载体背景下研究了多顺反子 miR，并评估了针对相同种子序列的多 miR 的基因敲低效力，同时针对报告基因和内源性 mRNA 靶标评估了针对不同种子序列的 miR 的基因敲低效力。我们证明，使用编码基于 miR 的单短发夹 RNA 的病毒载体可以在体外和体内获得有效的基因敲低，并报告了一种通用且有效的基于人工 miR30 的短发夹 RNA 的克隆平台，以生成有效的基因敲低病毒载体。
