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

  • Isabel Siegert
  • Valentin Schatz
  • Alexander T. Prechtel
  • Alexander Steinkasserer
  • Christian Bogdan
  • Jonathan Jantsch
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1121)

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.

Key words

siRNA Electroporation Dendritic cells Macrophages Gene expression 

References

  1. 1.
    Pei Y, Tuschl T (2006) On the art of identifying effective and specific siRNA. Nat Methods 3:670–676PubMedCrossRefGoogle Scholar
  2. 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 211:79–86PubMedCrossRefGoogle Scholar
  3. 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 166:7104–7111PubMedGoogle Scholar
  4. 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 171:691–696PubMedGoogle Scholar
  5. 5.
    Leon-Ponte M, Kirchhof MG, Sun T et al (2005) Polycationic lipids inhibit the pro-inflammatory response to LPS. Immunol Lett 96:73–83PubMedCrossRefGoogle Scholar
  6. 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 348:1079–1090PubMedCrossRefGoogle Scholar
  7. 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 337:71–77PubMedCrossRefGoogle Scholar
  8. 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 311:139–152PubMedCrossRefGoogle Scholar
  9. 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 353:102–110PubMedCrossRefGoogle Scholar
  10. 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 180:4697–4705PubMedGoogle Scholar
  11. 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 15:545–552PubMedCrossRefGoogle Scholar
  12. 12.
    Weintz G, Olsen JV, Fruhauf K et al (2010) The phosphoproteome of toll-like receptor-activated macrophages. Mol Syst Biol 6:371PubMedCentralPubMedCrossRefGoogle Scholar
  13. 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 107:18997–19001PubMedCentralPubMedCrossRefGoogle Scholar
  14. 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 90:551–562PubMedCrossRefGoogle Scholar
  15. 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):1455–1466PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Schleicher U, Bogdan C (2009) Generation, culture and flow-cytometric characterization of primary mouse macrophages. Methods Mol Biol 531:203–224PubMedCrossRefGoogle Scholar
  17. 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 223:77–92PubMedCrossRefGoogle Scholar
  18. 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 11:263–270PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Isabel Siegert
    • 1
  • Valentin Schatz
    • 1
  • Alexander T. Prechtel
    • 2
  • Alexander Steinkasserer
    • 3
  • Christian Bogdan
    • 1
  • Jonathan Jantsch
    • 1
  1. 1.Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und HygieneUniversitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-NürnbergErlangenGermany
  2. 2.Boehringer Ingelheim Pharma GmbH und Co. KGBiberach an der RißGermany
  3. 3.Abteilung Immunmodulation, HautklinikUniversitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen- NürnbergErlangenGermany

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