Skip to main content

Electroporation of siRNA into Mouse Bone Marrow-Derived Macrophages and Dendritic Cells

  • Protocol
  • First Online:
Electroporation Protocols

Abstract

Dendritic cells (DC) and macrophages (MΦ) play a pivotal role in antimicrobial defense, in the regulation of immune responses, and in maintaining tissue homeostasis. The analysis of DC and MΦ function relies on primary cells albeit these cells are known to be difficult to transfect. This makes the use of small interfering RNA (siRNA) for targeted manipulation of gene expression by RNA interference difficult. In the following chapter, we provide a detailed protocol for the successful transfer of siRNA via electroporation into a defined population of mouse bone marrow-derived MΦ or DC that does not cause toxicity to the myeloid cells or nonspecific alterations of their biological functions. Factors that influence the transfection and knockdown rate will be highlighted.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Pei Y, Tuschl T (2006) On the art of identifying effective and specific siRNA. Nat Methods 3:670–676

    Article  CAS  PubMed  Google Scholar 

  2. Mack KD, Wei R, Elbagarri A, Abbey N, McGrath MS (1998) A novel method for deae-dextran mediated transfection of adherent primary cultured human macrophages. J Immunol Methods 211:79–86

    Article  CAS  PubMed  Google Scholar 

  3. Zhang X, Wang JM, Gong WH, Mukaida N, Young HA (2001) Differential regulation of chemokine gene expression by 15-deoxy-delta 12,14 prostaglandin j2. J Immunol 166:7104–7111

    CAS  PubMed  Google Scholar 

  4. Hill JA, Ichim TE, Kusznieruk KP et al (2003) Immune modulation by silencing IL-12 production in dendritic cells using small interfering RNA. J Immunol 171:691–696

    CAS  PubMed  Google Scholar 

  5. Leon-Ponte M, Kirchhof MG, Sun T et al (2005) Polycationic lipids inhibit the pro-inflammatory response to LPS. Immunol Lett 96:73–83

    Article  CAS  PubMed  Google Scholar 

  6. Sioud M (2005) Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNA is sequence-dependent and requires endosomal localization. J Mol Biol 348:1079–1090

    Article  CAS  PubMed  Google Scholar 

  7. Jantsch J, Turza N, Volke M et al (2008) Small interfering RNA (siRNA) delivery into murine bone marrow-derived dendritic cells by electroporation. J Immunol Methods 337:71–77

    Article  CAS  PubMed  Google Scholar 

  8. Prechtel AT, Turza NM, Theodoridis AA, Kummer M, Steinkasserer A (2006) Small interfering RNA (siRNA) delivery into monocyte-derived dendritic cells by electroporation. J Immunol Methods 311:139–152

    Article  CAS  PubMed  Google Scholar 

  9. Wiese M, Castiglione K, Hensel M, Schleicher U, Bogdan C, Jantsch J (2010) Small interfering RNA (siRNA) delivery into murine bone marrow-derived macrophages by electroporation. J Immunol Methods 353:102–110

    Article  CAS  PubMed  Google Scholar 

  10. Jantsch J, Chakravortty D, Turza N et al (2008) Hypoxia and hypoxia-inducible factor-1 alpha modulate lipopolysaccharide-induced dendritic cell activation and function. J Immunol 180:4697–4705

    CAS  PubMed  Google Scholar 

  11. Machnik A, Neuhofer W, Jantsch J et al (2009) Macrophages regulate salt-dependent volume and blood pressure by a vascular endothelial growth factor-c-dependent buffering mechanism. Nat Med 15:545–552

    Article  CAS  PubMed  Google Scholar 

  12. Weintz G, Olsen JV, Fruhauf K et al (2010) The phosphoproteome of toll-like receptor-activated macrophages. Mol Syst Biol 6:371

    Article  PubMed Central  PubMed  Google Scholar 

  13. Luhrmann A, Nogueira CV, Carey KL, Roy CR (2010) Inhibition of pathogen-induced apoptosis by a Coxiella burnetii type iv effector protein. Proc Natl Acad Sci U S A 107:18997–19001

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Jantsch J, Wiese M, Schodel J et al (2011) Toll-like receptor activation and hypoxia use distinct signaling pathways to stabilize hypoxia-inducible factor 1{alpha} (HIF1A) and result in differential hif1a-dependent gene expression. J Leukoc Biol 90:551–562

    Article  CAS  PubMed  Google Scholar 

  15. Wiese M, Gerlach RG, Popp I et al (2012) Hypoxia-mediated impairment of the mitochondrial respiratory chain inhibits the bactericidal activity of macrophages. Infect Immun 80(4):1455–1466

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Schleicher U, Bogdan C (2009) Generation, culture and flow-cytometric characterization of primary mouse macrophages. Methods Mol Biol 531:203–224

    Article  CAS  PubMed  Google Scholar 

  17. Lutz MB, Kukutsch N, Ogilvie AL et al (1999) An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods 223:77–92

    Article  CAS  PubMed  Google Scholar 

  18. Hornung V, Guenthner-Biller M, Bourquin C et al (2005) Sequence-specific potent induction of ifn-alpha by short interfering rna in plasmacytoid dendritic cells through TLR7. Nat Med 11:263–270

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Siegert, I., Schatz, V., Prechtel, A.T., Steinkasserer, A., Bogdan, C., Jantsch, J. (2014). Electroporation of siRNA into Mouse Bone Marrow-Derived Macrophages and Dendritic Cells. In: Li, S., Cutrera, J., Heller, R., Teissie, J. (eds) Electroporation Protocols. Methods in Molecular Biology, vol 1121. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4614-9632-8_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4614-9632-8_9

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4614-9631-1

  • Online ISBN: 978-1-4614-9632-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics