HOMEPRODUCTSCOMPANYCONTACTFAQResearchDictionaryPharmaSign Up FREE or Login

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.
AuthorsBarbara Ahlemeyer, Julia-Franziska Vogt, Vera Michel, Petra Hahn-Kohlberger, Eveline Baumgart-Vogt
JournalHistochemistry and cell biology (Histochem Cell Biol) Vol. 142 Issue 5 Pg. 577-91 (Nov 2014) ISSN: 1432-119X [Electronic] Germany
PMID25224142 (Publication Type: Journal Article)
Chemical References
  • Fluorescent Dyes
  • PEX5 protein, human
  • Peroxisome-Targeting Signal 1 Receptor
  • Pex5 protein, mouse
  • RNA, Messenger
  • RNA, Small Interfering
  • Reactive Oxygen Species
  • Receptors, Cytoplasmic and Nuclear
  • dihydroethidine
  • Dicarbethoxydihydrocollidine
Topics
  • Dicarbethoxydihydrocollidine (analogs & derivatives, metabolism)
  • Electroporation (methods)
  • Fluorescent Dyes (metabolism)
  • Gene Knockdown Techniques
  • Hep G2 Cells
  • Humans
  • Peroxisomal Disorders (genetics, metabolism, pathology)
  • Peroxisome-Targeting Signal 1 Receptor
  • RNA, Messenger (analysis, antagonists & inhibitors, genetics)
  • RNA, Small Interfering (genetics)
  • Reactive Oxygen Species (metabolism)
  • Receptors, Cytoplasmic and Nuclear (analysis, biosynthesis, deficiency, genetics)
  • Transfection (methods)

Join CureHunter, for free Research Interface BASIC access!

Take advantage of free CureHunter research engine access to explore the best drug and treatment options for any disease. Find out why thousands of doctors, pharma researchers and patient activists around the world use CureHunter every day.
Realize the full power of the drug-disease research graph!


Choose Username:
Email:
Password:
Verify Password:
Enter Code Shown: