Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
PLoS One. 2011;6(7):e22437. doi: 10.1371/journal.pone.0022437. Epub 2011 Jul 18.
The use of RNAi in both basic and translational research often requires expression of multiple siRNAs from the same vector.
METHODS/PRINCIPAL FINDINGS: We have developed a novel chicken miR126-based artificial miRNA expression system that can express one, two or three miRNAs from a single cassette in a lentiviral vector. We show that each of the miRNAs expressed from the same lentiviral vector is capable of potent inhibition of reporter gene expression in transient transfection and stable integration assays in chicken fibroblast DF-1 cells. Transduction of Vero cells with lentivirus expressing two or three different anti-influenza miRNAs leads to inhibition of influenza virus production. In addition, the chicken miR126-based expression system effectively inhibits reporter gene expression in human, monkey, dog and mouse cells. These results demonstrate that the flanking regions of a single primary miRNA can support processing of three different stem-loops in a single vector.
CONCLUSIONS/SIGNIFICANCE: This novel design expands the means to express multiple miRNAs from the same vector for potent and effective silencing of target genes and influenza virus.
RNAi 在基础和转化研究中的应用通常需要从同一个载体中表达多个 siRNA。
方法/主要发现：我们开发了一种新型基于鸡 miR126 的人工 miRNA 表达系统，该系统可在慢病毒载体中单一盒式中表达一个、两个或三个 miRNA。我们表明，从同一个慢病毒载体表达的每个 miRNA 都能够在鸡成纤维细胞 DF-1 中的瞬时转染和稳定整合测定中有效抑制报告基因的表达。用表达两种或三种不同抗流感 miRNA 的慢病毒转导 Vero 细胞会导致流感病毒产量的抑制。此外，基于鸡 miR126 的表达系统可有效抑制人、猴、狗和鼠细胞中的报告基因表达。这些结果表明，单个初级 miRNA 的侧翼区域可支持在单个载体中处理三个不同的茎环。
结论/意义：这种新型设计扩展了从同一个载体中表达多个 miRNA 的手段，可有效沉默靶基因和流感病毒。
