A simple and robust vector-based shRNA expression system used for RNA interference

PLoS One. 2013;8(2):e56110. doi: 10.1371/journal.pone.0056110. Epub 2013 Feb 6.

Abstract

Background: RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive.

Results: In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo.

Conclusion: This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Carcinoma, Hepatocellular / genetics
  • Carcinoma, Hepatocellular / virology
  • Gene Expression Regulation, Viral
  • Gene Silencing*
  • Genetic Engineering
  • Genetic Vectors*
  • Hepatitis B Surface Antigens / chemistry
  • Hepatitis B Surface Antigens / genetics*
  • Hepatitis B Surface Antigens / metabolism
  • Hepatitis B e Antigens / chemistry
  • Hepatitis B e Antigens / genetics*
  • Hepatitis B e Antigens / metabolism
  • Hepatitis B virus / genetics*
  • Humans
  • Liver Neoplasms / genetics
  • Liver Neoplasms / virology
  • Mice
  • Mice, Inbred C57BL
  • Molecular Sequence Data
  • RNA Interference*
  • RNA, Small Interfering / genetics*
  • Tumor Cells, Cultured

Substances

  • Hepatitis B Surface Antigens
  • Hepatitis B e Antigens
  • RNA, Small Interfering

Grants and funding

This work was financially supported by the National Natural Science Foundation of China 81202562 and 81230089, and the Chinese Science and Technology Key Projects grants 2012ZX10004503-011 and 2012ZX09103301-044. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.