title: Two approaches to construct mammalian expression vector of shRNA to reduce expression and replication of HBV in vitro.
abstract: Two approaches have been developed to construct plasmids that mediate RNA interference to inhibit the replication and expression of HBV in 2.2.15 cell. The overlapping PCR extension and restriction enzyme-digestion were used to generate DNA fragments encoding designed shRNA based on sequences of ORF C of HBV genome. The pU6 derived vectors were constructed to develop plasmid based shRNA delivery systems termed pU6/HBVi. There were significant reductions in the expression of HBsAg and HBeAg between cells transfected with pU6/HBVi and control groups (as to HBsAg: P < 0. 01; and HBeAg: P < 0. 01). Consistently, the HBV DNA copies were reduced from 2.71 x 10(7) to <5 x 10(2) copies with or without pU6/HBVi. These results suggested that shRNA delivery by recombinant plasmids harboring shRNA encoding DNA fragment of interest generated either by overlapping PCR extension or restriction enzyme-digestion, could inhibit expressions of viral proteins and reduce viral replications. The pU6 derived plasmids might be a useful shRNA delivery system in mammalian cells. In addition, we found siRNA based on stealth 2311 was a potent RNAi target of HBV genome.
authors.affiliation: Department of Medical Research, Guangzhou Liuhuaqiao Hospital, Liuhua Road 111, Guangzhou, Guangdong Province, P. R. China.
authors.forename: Hong-Bin
authors.initials: HB
authors.lastname: Zhang
authors.forename: Jie
authors.initials: J
authors.lastname: Wu
authors.forename: Jiang
authors.initials: J
authors.lastname: Xian
authors.forename: Lei
authors.initials: L
authors.lastname: Pei
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
mesh_terms: D045744:Cell Line, Tumor; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006515:Hepatitis B virus / Q000502:physiology*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D014779:Virus Replication
chemical_list: D034741:RNA, Small Interfering