title: Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation.
abstract: The manipulation of the RNA interference pathway using small interfering RNA (siRNA) has become the most frequently used gene silencing method. However, siRNA delivery into primary cells, especially primary macrophages, is often considered challenging. Here we report the investigation of the suitability of two methodologies: transient transfection and electroporation, to deliver siRNA targeted against the putative immunomodulatory gene Mediterranean fever (MEFV) into primary bovine monocyte-derived macrophages (bMDM). Eleven commercial transfection reagents were investigated with variable results with respect to siRNA uptake, target gene knock-down, cell toxicity and type I interferon (IFN) response induction. Three transfection reagents: Lipofectamine 2000, Lipofectamine RNAiMAX and DharmaFECT 3, were found to consistently give the best results. However, all the transfection reagents tested induced an IFN response in the absence of siRNA, which could be minimized by reducing the transfection reagent incubation period. In addition, optimized siRNA delivery into bMDM by electroporation achieved comparable levels of target gene knock-down as transient transfection, without a detectable IFN response, but with higher levels of cell toxicity. The optimized transient transfection and electroporation methodologies may provide a starting point for optimizing siRNA delivery into macrophages derived from other species or other cells considered difficult to investigate with siRNA.
authors.affiliation: Division of Infection & Immunity, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian EH25 9RG, UK. Electronic address: Kirsty.Jensen@roslin.ed.ac.uk.
authors.forename: Kirsty
authors.initials: K
authors.lastname: Jensen
authors.affiliation: Division of Infection & Immunity, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian EH25 9RG, UK.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Anderson
authors.affiliation: Division of Infection & Immunity, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian EH25 9RG, UK.
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Glass
mesh_terms: D000818:Animals; D002417:Cattle; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000276:immunology; D018274:Electroporation / Q000662:veterinary*; D005260:Female; D055785:Gene Knockdown Techniques; D018390:Gene Targeting / Q000662:veterinary; D008264:Macrophages / Q000276:immunology*; D000071198:Pyrin; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics*; D014162:Transfection / Q000662:veterinary*
chemical_list: D003598:Cytoskeletal Proteins; D000071198:Pyrin; D034741:RNA, Small Interfering
keywords: Bovine; Electroporation; Macrophage; Transfection; siRNA