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Two approaches to construct mammalian expression vector of shRNA to reduce expression and replication of HBV in vitro

(2008) MOLECULAR BIOLOGY REPORTS. 35(3). p.465-472
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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.
Keywords
MICE, BIOLOGY, THERAPY, SUPPRESSION, GENE, INHIBITION, HBV, shRNA expression, plasmid construction, SHORT-HAIRPIN, HEPATITIS-B-VIRUS, SMALL INTERFERING RNAS

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Chicago
Zhang, Hong-Bin, Jie Wu, Jiang Xian, Lei Pei, and Jie Wang. 2008. “Two Approaches to Construct Mammalian Expression Vector of shRNA to Reduce Expression and Replication of HBV in Vitro.” Molecular Biology Reports 35 (3): 465–472.
APA
Zhang, H.-B., Wu, J., Xian, J., Pei, L., & Wang, J. (2008). Two approaches to construct mammalian expression vector of shRNA to reduce expression and replication of HBV in vitro. MOLECULAR BIOLOGY REPORTS, 35(3), 465–472.
Vancouver
1.
Zhang H-B, Wu J, Xian J, Pei L, Wang J. Two approaches to construct mammalian expression vector of shRNA to reduce expression and replication of HBV in vitro. MOLECULAR BIOLOGY REPORTS. 2008;35(3):465–72.
MLA
Zhang, Hong-Bin, Jie Wu, Jiang Xian, et al. “Two Approaches to Construct Mammalian Expression Vector of shRNA to Reduce Expression and Replication of HBV in Vitro.” MOLECULAR BIOLOGY REPORTS 35.3 (2008): 465–472. Print.
@article{746171,
  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 {\textlangle} 0. 01; and HBeAg: P {\textlangle} 0. 01). Consistently, the HBV DNA copies were reduced from 2.71 x 10(7) to {\textlangle} 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.},
  author       = {Zhang, Hong-Bin and Wu, Jie and Xian, Jiang and Pei, Lei and Wang, Jie},
  issn         = {0301-4851},
  journal      = {MOLECULAR BIOLOGY REPORTS},
  language     = {eng},
  number       = {3},
  pages        = {465--472},
  title        = {Two approaches to construct mammalian expression vector of shRNA to reduce expression and replication of HBV in vitro},
  url          = {http://dx.doi.org/10.1007/s11033-007-9108-0},
  volume       = {35},
  year         = {2008},
}

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