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

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.

SUBMITTER: Aznan AN

PROVIDER: S-EPMC6006497 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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

Aznan Afiah Nasuha AN Abdul Karim Norwahidah N Wan Ngah Wan Zurinah WZ Jubri Zakiah Z

Oncology letters 20180507 1

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 p ...[more]

PMID: 29930713

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

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

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