title: An efficient method for gene silencing in human primary plasmacytoid dendritic cells: silencing of the TLR7/IRF-7 pathway as a proof of concept.
abstract: Plasmacytoid dendritic cells (pDC) are specialized immune cells that produce massive levels of type I interferon in response to pathogens. Unfortunately, pDC are fragile and extremely rare, rendering their functional study a tough challenge. However, because of their central role in numerous pathologies, there is a considerable need for an efficient and reproducible protocol for gene silencing in these cells. In this report, we tested six different methods for siRNA delivery into primary human pDC including viral-based, lipid-based, electroporation, and poly-ethylenimine (PEI) technologies. We show that lipid-based reagent DOTAP was extremely efficient for siRNA delivery into pDC, and did not induce cell death or pDC activation. We successfully silenced Toll-Like Receptor 7 (TLR7), CXCR4 and IFN regulatory factor 7 (IRF-7) gene expression in pDC as assessed by RT-qPCR or cytometry. Finally, we showed that TLR7 or IRF-7 silencing in pDC specifically suppressed IFN-α production upon stimulation, providing a functional validation of our transfection protocol.
authors.affiliation: Equipe Chimie et Biologie, Modélisation &Immunologie pour la Thérapie (CBMIT), CNRS UMR8601, Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, CICB-Paris (FR 3567), Centre Universitaire des Saints-Pères, 45 rue des Saints Pères, 75006, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Nikaïa
authors.initials: N
authors.lastname: Smith
authors.affiliation: Equipe Chimie et Biologie, Modélisation &Immunologie pour la Thérapie (CBMIT), CNRS UMR8601, Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, CICB-Paris (FR 3567), Centre Universitaire des Saints-Pères, 45 rue des Saints Pères, 75006, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Vidalain
authors.affiliation: Université Paris Descartes, Sorbonne Paris Cité, Paris, France.|INSERM UMR-S 1124, 45 rue des Saints-Pères, 75006 Paris, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Nisole
authors.affiliation: Equipe Chimie et Biologie, Modélisation &Immunologie pour la Thérapie (CBMIT), CNRS UMR8601, Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, CICB-Paris (FR 3567), Centre Universitaire des Saints-Pères, 45 rue des Saints Pères, 75006, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Herbeuval
mesh_terms: D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism*; D018274:Electroporation / Q000379:methods; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry*; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D050839:Interferon Regulatory Factor-7 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016898:Interferon-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D011094:Polyethyleneimine / Q000737:chemistry; D061251:Primary Cell Culture; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051199:Toll-Like Receptor 7 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: C527276:CXCR4 protein, human; D005229:Fatty Acids, Monounsaturated; D050839:Interferon Regulatory Factor-7; D016898:Interferon-alpha; D000644:Quaternary Ammonium Compounds; D034741:RNA, Small Interfering; D019718:Receptors, CXCR4; C495348:TLR7 protein, human; D051199:Toll-Like Receptor 7; D011094:Polyethyleneimine; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane