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Use of RNA Interference to Investigate Cytokine Signal Transduction in Pancreatic Beta Cells

  • Fabrice MooreEmail author
  • Daniel A. Cunha
  • Hindrik Mulder
  • Decio L. Eizirik
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 820)

Abstract

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by immune infiltration of the pancreatic islets resulting in an inflammatory reaction named insulitis and subsequent beta cell apoptosis. During the course of insulitis beta cell death is probably caused by direct contact with activated macrophages and T-cells, and/or exposure to soluble mediators secreted by these cells, including cytokines, nitric oxide, and free oxygen radicals. In vitro exposure of beta cells to the cytokines interleukin(IL)-1β + interferon(IFN)-γ or to tumor necrosis factor(TNF)-α + IFN-γ induces beta cell dysfunction and ultimately apoptosis. The transcription factors NF-κB and STAT1 are key regulators of cytokine-induced beta cell death. However, little is known about the gene networks regulated by these (or other) transcription factors that trigger beta cell apoptosis. The recent development of RNA interference (RNAi) technology offers a unique opportunity to decipher the cytokine-activated molecular pathways responsible for beta cell death. Use of RNAi has been hampered by technical difficulties in transfecting primary beta cells, but in recent years we have succeeded in developing reliable and reproducible protocols for RNAi in beta cells. This chapter details the methods and settings used to achieve efficient and nontoxic transfection of small interfering RNA in immortal and primary beta cells.

Key words

Small interfering RNA siRNA Pancreatic beta cells Apoptosis Gene knockdown Inducible nitric oxide synthase Interleukin-1β Interferon-γ Tumor necrosis factor-α 

Notes

Acknowledgments

This work has been supported by grants from the Fonds National de la Recherche Scientifique (FNRS – FRSM) Belgium, the Communauté Française de Belgique – Actions de Recherche Concertées (ARC), the European Union (STREP Savebeta, contract no. 036903; in the Framework Programme 6 of the European Community) and the Belgium Program on Interuniversity Poles of Attraction initiated by the Belgium State (IUAP P6/40). F.M. is the recipient of a Post-Doctoral Fellowship from FNRS, Belgium. The authors have no duality of interest associated with this manuscript. We thank M.A. Neef, G. Vandenbroeck, M. Urbain, J. Schoonheydt, R. Leeman, A. M. Musuaya, and S. Mertens from the Laboratory of Experimental Medicine, ULB, for excellent technical support, Dr. Fernanda Ortis (Laboratory of Experimental Medicine) for helpful comments and Dr. Piero Marchetti (Department of Endocrinology and Metabolism, Metabolic Unit, University of Pisa, Pisa, Italy) for providing the human islets used for siRNA testing.

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Fabrice Moore
    • 1
    Email author
  • Daniel A. Cunha
    • 1
  • Hindrik Mulder
    • 2
  • Decio L. Eizirik
    • 1
  1. 1.Laboratory of Experimental MedicineUniversité Libre de BruxellesBrusselsBelgium
  2. 2.Unit of Molecular Metabolism, Department of Clinical Sciences in MalmöLund University Diabetes Center, Clinical Research Center 91:12MalmöSweden

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