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Generation of Innate Immune Reporter Cells Using Retroviral Transduction

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Innate Immune Activation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1714))

Abstract

Innate immune cells are notoriously difficult to transfect; however, retroviruses can be used to stably integrate genes of interest into the host genome of primary or immortalized immune cells resulting in the generation of reporter cells. Here, we provide a detailed protocol covering the production of retroviruses, retroviral infection of innate immune target cells (including isolation and differentiation of murine bone marrow cells to macrophages), and several methods for enrichment of positively transduced cells.

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References

  1. Labzin LI, Schmidt SV, Masters SL, Beyer M, Krebs W, Klee K, Stahl R, Lutjohann D, Schultze JL, Latz E, De Nardo D (2015) ATF3 is a key regulator of macrophage IFN responses. J Immunol 195(9):4446–4455

    Article  CAS  PubMed  Google Scholar 

  2. Franklin BS, Bossaller L, De Nardo D, Ratter JM, Stutz A, Engels G, Brenker C, Nordhoff M, Mirandola SR, Al-Amoudi A, Mangan MS, Zimmer S, Monks BG, Fricke M, Schmidt RE, Espevik T, Jones B, Jarnicki AG, Hansbro PM, Busto P, Marshak-Rothstein A, Hornemann S, Aguzzi A, Kastenmuller W, Latz E (2014) The adaptor ASC has extracellular and ‘prionoid’ activities that propagate inflammation. Nat Immunol 15(8):727–737

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Sun J, Li N, KS O, Dutta B, Vayttaden SJ, Lin B, Ebert TS, De Nardo D, Davis J, Bagirzadeh R, Lounsbury NW, Pasare C, Latz E, Hornung V, Fraser ID (2016) Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use. Sci Signal 9(409):ra3

    Article  PubMed  PubMed Central  Google Scholar 

  4. Beilharz M, De Nardo D, Latz E, Franklin BS (2016) Measuring NLR oligomerization II: detection of ASC speck formation by confocal microscopy and immunofluorescence. Methods Mol Biol 1417:145–158

    Article  CAS  PubMed  Google Scholar 

  5. De Nardo D (2015) Toll-like receptors: activation, signalling and transcriptional modulation. Cytokine 74(2):181–189

    Article  PubMed  Google Scholar 

  6. Motshwene PG, Moncrieffe MC, Grossmann JG, Kao C, Ayaluru M, Sandercock AM, Robinson CV, Latz E, Gay NJ (2009) An oligomeric signaling platform formed by the toll-like receptor signal transducers MyD88 and IRAK-4. J Biol Chem 284(37):25404–25411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lin SC, Lo YC, Wu H (2010) Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling. Nature 465(7300):885–890

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ferrao R, Zhou H, Shan Y, Liu Q, Li Q, Shaw DE, Li X, Wu H (2014) IRAK4 dimerization and trans-autophosphorylation are induced by Myddosome assembly. Mol Cell 55(6):891–903

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Suzuki N, Suzuki S, Duncan GS, Millar DG, Wada T, Mirtsos C, Takada H, Wakeham A, Itie A, Li S, Penninger JM, Wesche H, Ohashi PS, Mak TW, Yeh WC (2002) Severe impairment of interleukin-1 and toll-like receptor signalling in mice lacking IRAK-4. Nature 416(6882):750–756

    Article  CAS  PubMed  Google Scholar 

  10. Picard C, Puel A, Bonnet M, Ku CL, Bustamante J, Yang K, Soudais C, Dupuis S, Feinberg J, Fieschi C, Elbim C, Hitchcock R, Lammas D, Davies G, Al-Ghonaium A, Al-Rayes H, Al-Jumaah S, Al-Hajjar S, Al-Mohsen IZ, Frayha HH, Rucker R, Hawn TR, Aderem A, Tufenkeji H, Haraguchi S, Day NK, Good RA, Gougerot-Pocidalo MA, Ozinsky A, Casanova JL (2003) Pyogenic bacterial infections in humans with IRAK-4 deficiency. Science 299(5615):2076–2079

    Article  CAS  PubMed  Google Scholar 

  11. Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, Fitzgerald KA, Latz E (2008) Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 9(8):847–856

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kutner RH, Zhang XY, Reiser J (2009) Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc 4(4):495–505

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank R. Stahl (Institute of Innate Immunity, Bonn) for assistance in generating the murine IRAK4-mCitrine and control mCitrine retroviral plasmids. This work was supported in part by the intramural BONFOR research support at the University of Bonn (D.D).

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Authors and Affiliations

  1. Department of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076, Tübingen, Germany

    Yamel Cardona Gloria

  2. Institute of Innate Immunity, University Hospital, University of Bonn, Sigmund Freud Str. 25, 53127, Bonn, Germany

    Eicke Latz

  3. Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA, 01605, USA

    Eicke Latz

  4. German Center for Neurodegenerative Diseases, Bonn, 53175, Germany

    Eicke Latz

  5. Inflammation Division, The Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Dominic De Nardo

  6. Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia

    Dominic De Nardo

Authors
  1. Yamel Cardona Gloria
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  2. Eicke Latz
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  3. Dominic De Nardo
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Corresponding author

Correspondence to Dominic De Nardo .

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Editors and Affiliations

  1. Inflammation Division, The Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Dominic De Nardo

  2. Chemical Biology Division, The Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Christine M De Nardo

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Cardona Gloria, Y., Latz, E., De Nardo, D. (2018). Generation of Innate Immune Reporter Cells Using Retroviral Transduction. In: De Nardo, D., De Nardo, C. (eds) Innate Immune Activation. Methods in Molecular Biology, vol 1714. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7519-8_7

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  • DOI: https://doi.org/10.1007/978-1-4939-7519-8_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7518-1

  • Online ISBN: 978-1-4939-7519-8

  • eBook Packages: Springer Protocols

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