title: Microporation is an efficient method for siRNA-induced knockdown of PEX5 in HepG2 cells: evaluation of the transfection efficiency, the PEX5 mRNA and protein levels and induction of peroxisomal deficiency.
abstract: The pathomechanism of peroxisomal biogenesis disorders (PBDs), a group of inherited autosomal recessive diseases with mutations of peroxin (PEX) genes, is not yet fully understood. Therefore, several knockout models, e.g., the PEX5 knockout mouse, have been generated exhibiting a complete loss of peroxisomal function. In this study, we wanted to knockdown PEX5 using the siRNA technology (1) to mimic milder forms of PBDs in which the mutated peroxin has some residual function and (2) to analyze the cellular consequences of a reduction of the PEX5 protein without adaption during the development as it is the case in a knockout animal. First, we tried to optimize the transfection of the hepatoma cell line HepG2 with PEX5 siRNA using different commercially available liposomal and non-liposomal transfection reagents (Lipofectamine(®) 2000, FuGENE 6, HiPerFect(®), INTERFERin™, RiboJuice™) as well as microporation using the Neon™ Transfection system. Microporation was found to be superior to the transfection reagents with respect to the transfection efficiency (100 vs. 0-70%), to the reduction of PEX5 mRNA (by 90 vs. 0-50%) and PEX5 protein levels (by 70 vs. 0-50%). Interestingly, we detected that a part of the cleaved PEX5 mRNA still existed as 3' fragment (15%) 24 h after microporation. Using microporation, we further analyzed whether the reduced PEX5 protein level impaired peroxisomal function. We indeed detected a reduced targeting of SKL-tagged proteins into peroxisomes as well as an increased oxidative stress as found in PBD patients and respective knockout mouse models. Knockdown of the PEX5 protein and functional consequences were at a maximum 48 h after microporation. Thereafter, the PEX5 protein was resynthesized, which may allow the temporal analysis of the loss as well as the reconstitution of peroxisomes in the future. In conclusion, we propose microporation as an efficient and reproducible method to transfect HepG2 cells with PEX5 siRNA. We succeeded to transiently knockdown PEX5 mRNA and its protein level leading to functional consequences similar as observed in peroxisome deficiencies.
authors.affiliation: Division of Medical Cell Biology, Institute for Anatomy and Cell Biology, Justus Liebig University, Aulweg 123, 35385, Giessen, Germany, Barbara.Ahlemeyer@anatomie.med.uni-giessen.de.
authors.forename: Barbara
authors.initials: B
authors.lastname: Ahlemeyer
authors.forename: Julia-Franziska
authors.initials: JF
authors.lastname: Vogt
authors.forename: Vera
authors.initials: V
authors.lastname: Michel
authors.forename: Petra
authors.initials: P
authors.lastname: Hahn-Kohlberger
authors.forename: Eveline
authors.initials: E
authors.lastname: Baumgart-Vogt
mesh_terms: D003997:Dicarbethoxydihydrocollidine / Q000031:analogs & derivatives / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D055785:Gene Knockdown Techniques; D056945:Hep G2 Cells; D006801:Humans; D018901:Peroxisomal Disorders / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000074435:Peroxisome-Targeting Signal 1 Receptor; D012333:RNA, Messenger / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis / Q000096:biosynthesis / Q000172:deficiency* / Q000235:genetics*; D014162:Transfection / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; C000614264:PEX5 protein, human; D000074435:Peroxisome-Targeting Signal 1 Receptor; C000614265:Pex5 protein, mouse; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D018160:Receptors, Cytoplasmic and Nuclear; C514241:dihydroethidine; D003997:Dicarbethoxydihydrocollidine