title: Characterization of the swine U6 promoter for short hairpin RNA expression and its application to inhibition of virus replication.
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.
authors.affiliation: Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan, ROC.
authors.forename: Ching-Wei
authors.initials: CW
authors.lastname: Wu
authors.forename: Maw-Sheng
authors.initials: MS
authors.lastname: Chien
authors.forename: Chienjin
authors.initials: C
authors.lastname: Huang
mesh_terms: D000818:Animals; D034741:RNA, Small Interfering / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics*; D013552:Swine; D014779:Virus Replication / Q000235:genetics / Q000502:physiology*
chemical_list: D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA
keywords: Classical swine fever virus; RNA interference; Short hairpin RNAs; U6 promoter