Optimized Protocols for siRNA Delivery into Monocytes and Dendritic Cells

  • Anne Mobergslien
  • Mouldy Sioud
Part of the Methods in Molecular Biology book series (MIMB, volume 629)


Over the past decade, immunotherapy has emerged as a promising alternative form of cancer treatment with the potential to eradicate tumour metastasis. Unfortunately, its curative potential is in general limited by immunosuppressive proteins that negatively regulate dendritic (DC) and/or T-cell function. The recent discovery of RNA interference (RNAi) has facilitated the study of gene function in immune cells and recent data indicate that DC maturation, function, and survival can be modulated by small interfering RNAs (siRNAs) targeting genes involved in immune suppression. This chapter describes detailed protocols for introducing siRNAs into human monocytes and dendritic cells using standard electroporation techniques.

Key words

RNA interference siRNAs dendritic cells electroporation 



This work was supported by the Gene therapy program at the Norwegian Radium Hospital to M. Sioud. We thank Dr. Anne Dybwad for editing the manuscript.


  1. 1.
    Grütz, G. (2005) New insights into the molecular mechanism of interleukin-10-mediated immunosuppression. J Leukoc Biol, 77, 3–15.PubMedGoogle Scholar
  2. 2.
    Munn, D.H. and Mellor, A.L. (2004) IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat Rev Immunol, 4, 762–774.PubMedCrossRefGoogle Scholar
  3. 3.
    Munn, D.H., Zhou, M., Attwood, J.T., Bondarev, I., Conway, S.J., Marshall, B., Brown, C., and Mellor, A.L. (1998) Prevention of allogeneic fetal rejection by tryptophan catabolism. Science, 281, 1191–1193.PubMedCrossRefGoogle Scholar
  4. 4.
    Munn, D.H., Sharma, M.D., Hou, D., Baban, B., Lee, J.R., Antonia, S.J., Messina, J.L., Chandler, P., Koni, P.A., and Mellor, A.L. (2004) Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes. J Clin Invest, 114, 280–290.PubMedGoogle Scholar
  5. 5.
    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.PubMedCrossRefGoogle Scholar
  6. 6.
    Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotides RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494–498.PubMedCrossRefGoogle Scholar
  7. 7.
    Sioud, M. (2004) Therapeutic siRNAs. Trends Pharmacol Sci, 25, 22–28.PubMedCrossRefGoogle Scholar
  8. 8.
    Behlke, M.A. (2006) Progress towards in vivo use of siRNAs. Mol Ther, 13, 644–670.PubMedCrossRefGoogle Scholar
  9. 9.
    Sioud, M. (2005) Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization. J Mol Biol, 348, 1079–1090.PubMedCrossRefGoogle Scholar
  10. 10.
    Hornung, V., Guenthner-Biller, M., Bourquin, C., Ablasser, A., Schlee, M., Uematsu, S., Noronha, A., Manoharan, M., Akira, S., de Fougerolles, A., Endres, S., and Hartmann, G. (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med, 11, 263–270.PubMedCrossRefGoogle Scholar
  11. 11.
    Judge, A.D., Sood, V., Shaw, J.R., Fang, D., McClintock, K., and MacLachlan, I. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol, 23, 457–462.PubMedCrossRefGoogle Scholar
  12. 12.
    Furset, G. and Sioud, M. (2007) Design of bifunctional siRNAs: combining immunostimulation and gene-silencing in one single siRNA molecule. Biochem Biophys Res Commun, 352, 642–649.PubMedCrossRefGoogle Scholar
  13. 13.
    Takeuchi, O. and Akira, S. (2007) Recognition of viruses by innate immunity. Immunol Rev, 220, 214–224.PubMedCrossRefGoogle Scholar
  14. 14.
    Watts, C. (2004) The bell tolls for phagosome maturation. Science, 304, 976–977.PubMedCrossRefGoogle Scholar
  15. 15.
    Sioud, M. (2008) Does understanding of immune activation by RNA predict the design of safe siRNAs. Front Biosci, 13, 4379–4392.PubMedCrossRefGoogle Scholar
  16. 16.
    Iversen, P.O., Semaeva, E., Sørensen, D.R., Wiig, H., and Sioud, M. (2009) Dendritic cells loaded with tumor antigens and a dual immunostimulatory and anti-interleukin-10-specific small interference RNA prime T lymphocytes against leukemic cells. Transl Oncol, 2, 242–246.Google Scholar
  17. 17.
    Flatekval, G.F. and Sioud, M. (2009) Modulation of dendritic cell maturation and function with mono- and bifunctional small interfering RNAs targeting indoleamine 2,3-dioxygenase. Immunology, 128, e837–e848. DOI: 10.1111/j.1365-2567.2009.03093.x.PubMedCrossRefGoogle Scholar
  18. 18.
    Van Tendeloo, V.F., Ponsaerts, P., Lardon, F., Nijs, G., Lenjou, M., Van Broeckhoven, C., Van Bockstaele, D.R., and Berneman, Z.N. (2001) Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood, 98, 49–56.PubMedCrossRefGoogle Scholar
  19. 19.
    Michiels, A., Tuyaerts, S., Bonehill, A., Corthals, J., Breckpot, K., Heirman, C., Van Meirvenne, S., Dullaers, M., Allard, S., Brasseur, F., van der Bruggen, P., and Thielemans, K. (2005) Electroporation of immature and mature dendritic cells: implications for dendritic cell-based vaccines. Gene Ther, 12, 772–782.PubMedCrossRefGoogle Scholar
  20. 20.
    Prechtel, A.T., Turza, N.M., Theodoridis, A.A., Kummer, M., and Steinkasserer, A. (2006) Small interfering RNA (siRNA) delivery into monocyte-derived dendritic cells by electroporation. J Immunol Methods, 311, 139–152.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Anne Mobergslien
    • 1
  • Mouldy Sioud
    • 1
  1. 1.Departments of ImmunologyInstitute for Cancer Research, Rikshospitalet-Radiumhospitalet, University HospitalOsloNorway

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