Critical factors for lentivirus-mediated PRDX4 gene transfer in the HepG2 cell line

Aznan, A.N.; Abdul Karim, N.; Wan Ngah, W.Z.; Jubri, Z.

Oncology Letters 16(1): 73-82

2018


ISSN/ISBN: 1792-1074
PMID: 29930713
DOI: 10.3892/ol.2018.8650
Accession: 057269691

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Abstract
Optimization of critical factors affects transduction efficiency and is able to reduce reagent consumption. The present study aimed to determine the optimum transduction conditions of small hairpin (sh)RNA against peroxiredoxin 4 (PRDX4) in the HepG2 cell line. Cell viability assays were conducted based on serum condition, incubation time, polybrene concentration and antibiotic dose selection. Non-targeting control shRNA was transduced into HepG2 cells in a 5-fold serial dilution, and colonies positive for green fluorescent protein were counted using ImageJ software. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to validate PRDX4 expression. The optimum cell density for transduction was 5.0×103 cells/well in 96-well plates to achieve 40 to 50% confluency the following day. The transduction media consisted of 10% fetal bovine serum (FBS) and 12 µg/ml polybrene, and was used to dilute lentiviral particles at a functional titer of 4.9×105 TU/ml for multiplicity of infection (MOI) of 20, 15 and 10, for 24 h of incubation. Selection with 7 µg/ml puromycin was performed in transduced cells. shRNA 3 was revealed to inhibit PRDX4 mRNA and protein expression. In conclusion, PRDX4 was successfully silenced in 5.0×103 HepG2 cells cultured with 10% FBS and 12 µg/ml polybrene, at a 4.9×105 TU/ml functional titer for MOI of 20, 15 and 10.