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.