title: A comparative study of transfection methods for RNA interference in bone marrow-derived murine dendritic cells.
abstract: Selective gene silencing using RNA interference (RNAi) has been shown to be an efficient method for manipulation of cellular functions. In this study, we compare three previously established methods for transfection of murine bone marrow-derived DC (BM-DC). We tested the efficacy of electroporation with the Mouse Nucleofector kit((R)) from Amaxa Biosystems and lipid-based transfection methods using transfection reagents from Santa Cruz Biotechnology or Genlantis. To analyse the transfection efficacy we used FITC-conjugated siRNA as a positive control together with CD80 and CD86 specific siRNA. We show that electroporation using the Mouse Nucleofector kit((R)) from Amaxa Biosystems was not an efficient method to transfect BM-DC with siRNA in our hands. Transfection with Santa Cruz Biotechnology reagents resulted in up to 59% FITC-siRNA positive cells, but did not result in effective silencing of CD80 surface expression. In contrast, the most effective method was the lipid-based method using the siRNA transfection reagent GeneSilencer((R)) from Genlantis. This protocol resulted in up to 92% FITC-siRNA positive cells after 4 h which declined to 62% and 59% 24 and 48 h post-transfection, respectively. The transfected BM-DC remained CD11c positive, expressed high MHC class II and intermediate CD40 and were functional as APC. In conclusion, this protocol was effective for manipulation of murine BM-DC function through the use of specific siRNA and such methods can be important for the future study of DC-T cell interactions.
authors.affiliation: Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen N, Denmark.
authors.forename: C D
authors.initials: CD
authors.lastname: Pedersen
authors.forename: J J
authors.initials: JJ
authors.lastname: Fang
authors.forename: A E
authors.initials: AE
authors.lastname: Pedersen
mesh_terms: D000818:Animals; D018122:B7-1 Antigen / Q000235:genetics; D051940:B7-2 Antigen / Q000235:genetics; D001854:Bone Marrow Cells / Q000502:physiology*; D003713:Dendritic Cells / Q000502:physiology*; D018274:Electroporation; D005260:Female; D005434:Flow Cytometry; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D034622:RNA Interference; D014162:Transfection / Q000379:methods*
chemical_list: D018122:B7-1 Antigen; D051940:B7-2 Antigen