Optimized Gene Silencing by Co-expression of Multiple shRNAs in a Single Vector

  • Yasuhito IshigakiEmail author
  • Akihiro Nagao
  • Tsukasa Matsunaga
Part of the Methods in Molecular Biology book series (MIMB, volume 623)


Currently, RNA interference technology is one of the most powerful tools in molecular biology and has been widely used in genetic manipulation. In addition to chemically synthesized small interfering RNA (siRNA), vector-based methods have been developed for stable gene silencing by the expression of a single short-hairpin RNA (shRNA). The artificially expressed RNA molecules are processed to form a silencing complex that causes the specific degradation of its target mRNA. However, silencing vectors containing a single shRNA-expressing sequence sometimes induce only poor knockdown. In order to improve the knockdown efficiency using shRNA, the multiple shRNA-expressing sequences were introduced into a single plasmid vector. Compared with the conventional single shRNA-expression vector, the multiple shRNA-expression vectors confer higher yields of stable clones with efficient knockdown and better correlations between knockdown level and the expression level of second marker gene, enhanced green fluorescent protein, in the vector. These features are very helpful for establishing stable knockdown clones and the detailed procedure is described in this chapter.

Key words

RNA interference Knockdown shRNA Plasmid vector XPA 



The authors thank Dr. Xia Zhao and Prof. H. Nakagawa of Kanazawa Medical University for his kind support. This work was also supported by KAKENHI, Kanazawa Medical University (S2007-3) and Kanazawa University.


  1. 1.
    Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.CrossRefPubMedGoogle Scholar
  2. 2.
    Williams, B.R. (1999) PKR; a sentinel kinase for cellular stress. Oncogene 18, 6112-6120.CrossRefPubMedGoogle Scholar
  3. 3.
    Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498.CrossRefPubMedGoogle Scholar
  4. 4.
    Elbashir, S.M., Lendeckel, W. and Tuschl, T. (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188-200.CrossRefPubMedGoogle Scholar
  5. 5.
    Yu, J.Y., DeRuiter, S.L. and Turner, D.L. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 99, 6047-6052.CrossRefPubMedGoogle Scholar
  6. 6.
    Brummelkamp, T.R., Bernards, R. and Agami, R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-553.CrossRefPubMedGoogle Scholar
  7. 7.
    Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J. and Conklin, D.S. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948-958.CrossRefPubMedGoogle Scholar
  8. 8.
    Zhou, H., Xia, X.G. and Xu, Z. (2005) An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi. Nucleic Acids Res. 33, e62.CrossRefPubMedGoogle Scholar
  9. 9.
    Tang, G. (2004) siRNA and miRNA:an insight into RISCs. Trends Biochem. Sci. 30,106-114.CrossRefGoogle Scholar
  10. 10.
    Nagao, A., Zhao, X., Takegami, T., Nakagawa, H., Matsu, S., Matsunaga, T., et al. (2008) Multiple shRNA expressions in a single plasmid vector improve RNAi against the XPA gene. Biochem. Biophys. Res. Commun. 370, 301-305.CrossRefPubMedGoogle Scholar
  11. 11.
    Kobayashi, T., Takeuchi, S., Saijo, M., Nakatsu, Morioka, Y.H., Otsuka, E., et al. (1998) Mutational analysis of a function of xeroderma pigmentosum group A (XPA) protein in strand-specific DNA repair. Nucleic Acids Res. 26, 4662-4668.Google Scholar
  12. 12.
    Satotaka, I., Tanaka, K., Miura, N., Miyamoto, I., Satoh, Y., Kondo, S., et al. (1990) Characterization of a splicing mutation in group A xeroderma pigmentosum. Proc. Natl. Acad. Sci. U.S.A. 87, 9908-9912.CrossRefGoogle Scholar
  13. 13.
    Chang, Y.F., Imam, J.S. and Wilkinson, M.F. (2007) The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem. 76, 51-74.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Yasuhito Ishigaki
    • 1
    Email author
  • Akihiro Nagao
    • 1
  • Tsukasa Matsunaga
    • 2
  1. 1.Division of Core FacilityMedical Research Institute, Kanazawa Medical UniversityKahoku-gunJapan
  2. 2.Laboratory of Human Molecular Genetics, Graduate School of Natural Science and TechnologyKanazawa UniversityKanazawaJapan

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