Two approaches to construct mammalian expression vector of shRNA to reduce expression and replication of HBV in vitro.
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.
Zhang
Hong-Bin
HB
Department of Medical Research, Guangzhou Liuhuaqiao Hospital, Liuhua Road 111, Guangzhou, Guangdong Province, P. R. China.
Wu
Jie
J
Xian
Jiang
J
Pei
Lei
L
Wang
Jie
J
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
D034741:RNA, Small Interfering