title: Vpx-Independent Lentiviral Transduction and shRNA-Mediated Protein Knock-Down in Monocyte-Derived Dendritic Cells.
abstract: The function of dendritic cells (DCs) in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. To date, efficient lentiviral transduction of DCs or their monocyte derived counterparts (MDDCs) required high multiplicity of infection (MOI) or the exposure to the HIV-2/SIV protein Vpx to degrade viral restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1). Here we present a Vpx-independent method for efficient (>95%) transduction of MDDCs at lower MOI. The protocol can be used both for ectopic gene expression and knock-down. Introducing shRNA targeting viral entry receptor CD4 and restriction factor SAMHD1 into MDDCs resulted in down-regulation of targeted proteins and, consequently, expected impact on HIV infection. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting.
authors.affiliation: Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, De Pintelaan 185, Gent, Belgium.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Witkowski
authors.affiliation: Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, De Pintelaan 185, Gent, Belgium.
authors.forename: Jolien
authors.initials: J
authors.lastname: Vermeire
authors.affiliation: Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, De Pintelaan 185, Gent, Belgium.
authors.forename: Alessia
authors.initials: A
authors.lastname: Landi
authors.affiliation: Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, De Pintelaan 185, Gent, Belgium.
authors.forename: Evelien
authors.initials: E
authors.lastname: Naessens
authors.affiliation: Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, De Pintelaan 185, Gent, Belgium.
authors.forename: Hanne
authors.initials: H
authors.lastname: Vanderstraeten
authors.affiliation: Virology lab, Department of Virology, Parasitology and Immunology, Ghent University, Salisburylaan 133 D1, Merelbeke, Belgium.
authors.forename: Hans
authors.initials: H
authors.lastname: Nauwynck
authors.affiliation: Immunology lab, Department of Virology, Parasitology and Immunology, Ghent University, Salisburylaan 133 D1, Merelbeke, Belgium.
authors.forename: Herman
authors.initials: H
authors.lastname: Favoreel
authors.affiliation: Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, De Pintelaan 185, Gent, Belgium.
authors.forename: Bruno
authors.initials: B
authors.lastname: Verhasselt
mesh_terms: D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D014161:Transduction, Genetic; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics
chemical_list: D034741:RNA, Small Interfering; C513059:VPX protein, Simian immunodeficiency virus; D054334:Viral Regulatory and Accessory Proteins