title: Use of RNA interference to investigate cytokine signal transduction in pancreatic beta cells.
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.
authors: Laboratory of Experimental Medicine, Université Libre de Bruxelles, Brussels, BE 1070, Belgium. fmoore@ulb.ac.be
authors: Fabrice
authors: F
authors: Moore
authors: Daniel A
authors: DA
authors: Cunha
authors: Hindrik
authors: H
authors: Mulder
authors: Decio L
authors: DL
authors: Eizirik
mesh_terms: D000818:Animals; D017209:Apoptosis; D016923:Cell Death; D002470:Cell Survival; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D003922:Diabetes Mellitus, Type 1 / Q000503:physiopathology; D055785:Gene Knockdown Techniques; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D007340:Insulinoma / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology / Q000473:pathology; D008297:Male; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D017208:Rats, Wistar; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D016207:Cytokines; D053583:Interleukin-1beta; D016328:NF-kappa B; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II