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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

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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.

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Abbreviations

AP:

Alkaline phosphatase

Bp:

Base pairs

MEM:

Minimum Essential Medium

Neg siRNA:

Silencer™ negative control siRNA

Nt:

Nucleotide(s)

PBD:

Peroxisomal biogenesis disorder

PBS:

Phosphate-buffered saline

PEX:

Peroxin (peroxisomal biogenesis protein)

PEX5:

Human peroxisomal biogenesis factor 5 protein

PEX5 :

Human peroxisomal biogenesis factor 5 gene

PEX5 split:

PEX5 PCR primer complementary to the PEX5 siRNA cleavage site

PEX5 3′ end:

PEX5 PCR primer complementary to the 3′ fragment of the PEX5 siRNA cleavage

PTS1:

Peroxisomal targeting signal 1

PTS2:

Peroxisomal targeting signal 2

RISC:

RNA-induced silencing complex

ROS:

Reactive oxygen species

RT-PCR:

Reverse transcriptase polymerase chain reaction

SKL:

Serine–lysine–leucine–COOH at the C-terminus

TBS:

Tris-buffered saline

TBST:

Tris-buffered saline plus 0.05 % Tween 20

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Acknowledgments

The authors would like to thank Georg Lüers, Saba Hadad and Srikanth Karnati for their recommendation to use the Neon™ Transfection System as well as Gabriele Thiele and Andrea Textor for excellent technical assistance.

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Author notes

  1. Julia-Franziska Vogt

    Present address: Klinik für Hals-Nasen-Ohren Heilkunde, Universitätsklinikum Marburg-Giessen, Klinikstr. 33, 35392, Giessen, Germany

  2. Vera Michel

    Present address: Division of Reproductive Medicine, Institute for Anatomy and Cell Biology, Justus Liebig University, Aulweg 123, 35385, Giessen, Germany

Authors and Affiliations

  1. Division of Medical Cell Biology, Institute for Anatomy and Cell Biology, Justus Liebig University, Aulweg 123, 35385, Giessen, Germany

    Barbara Ahlemeyer, Julia-Franziska Vogt, Vera Michel, Petra Hahn-Kohlberger & Eveline Baumgart-Vogt

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  1. Barbara Ahlemeyer
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Correspondence to Barbara Ahlemeyer or Eveline Baumgart-Vogt.

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Ahlemeyer, B., Vogt, JF., Michel, V. et al. 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. Histochem Cell Biol 142, 577–591 (2014). https://doi.org/10.1007/s00418-014-1254-6

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