猪 U6 启动子的特性及其在短发夹 RNA 表达和抑制病毒复制中的应用。

Characterization of the swine U6 promoter for short hairpin RNA expression and its application to inhibition of virus replication.

机构信息

Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.

出版信息

J Biotechnol. 2013 Oct 10;168(1):78-84. doi: 10.1016/j.jbiotec.2013.07.009. Epub 2013 Jul 31.

DOI:10.1016/j.jbiotec.2013.07.009

PMID:23916945

Abstract

Expression of short hairpin RNAs (shRNAs) by the RNA polymerase type III U6 promoter is an effective and widely used strategy for RNA interference (RNAi) which is a sequence-specific gene silencing mechanism. The U6 promoters from human, mouse, and swine were cloned, respectively for constructing various shRNA expression vectors. The transcription efficiency of each U6 promoter was analyzed for its activity to drive expression of shRNA targeting enhanced green fluorescent protein (EGFP) mRNA in different mammalian cells. All three U6 promoters were functional and the swine U6 promoter demonstrated the most efficient knockdown of EGFP synthesis in all these three species of cell lines including porcine kidney (PK-15), human embryonic kidney (HEK293T), and mouse fibroblast (LM) cells. Furthermore, the antiviral effect of shRNA targeting the classical swine fever virus (CSFV) NS5B driven by the swine U6 promoter was confirmed by the significant reduction of virus replication.

摘要

短发夹 RNA（shRNA）的表达由 RNA 聚合酶 III U6 启动子驱动是 RNA 干扰（RNAi）的一种有效且广泛应用的策略，它是一种序列特异性的基因沉默机制。分别克隆了来自人、鼠和猪的 U6 启动子，用于构建各种 shRNA 表达载体。分析了每个 U6 启动子的转录效率，以研究其活性，从而驱动靶向增强型绿色荧光蛋白（EGFP）mRNA 的 shRNA 在不同哺乳动物细胞中的表达。这三个 U6 启动子都是有功能的，猪 U6 启动子在包括猪肾（PK-15）、人胚肾（HEK293T）和小鼠成纤维细胞（LM）在内的这三种细胞系中，对 EGFP 合成的抑制效果最为显著。此外，由猪 U6 启动子驱动的针对经典猪瘟病毒（CSFV）NS5B 的 shRNA 的抗病毒作用通过病毒复制的显著减少得到了证实。