Construction of lentivirus-mediated mito-OGG1 gene overexpression and OGG1 gene knockdown model in 661W cells

Authors: Li Han,  Ma Wei,  Shi Kangpei,  Wang Lei,  Huang Hao,  Wu Wenbin,  Zhang Xiaotong,  Zhu Xiaobo
DOI: 10.3760/cma.j.issn.2095-0160.2019.05.006
Published 2019-05-10
Cite as Chin J Exp Ophthalmol, 2019,37(5): 348-356.

Abstract

Objective

To construct and authenticate the lentiviral-mediated overexpression of mouse mitochondrial-targeted-8-oxoguanine DNA-glycosylase 1 (mito-OGG1) gene and the lentiviral-mediated short hairpin RNA (shRNA) down-regulation of OGG1 gene expression model in 661W cells.

Methods

Constructed the target plasmids, including pLenti-EF1a-EGFP-P2A-Puro-CMV-Mito-OGG1-3Flag (pLenti-OGG1-GFP) and pLKD-CMV-G&PR-U6-shRNA (pLKD-shRNA).293T cells were used to obtain green fluorescent protein (GFP)-tagged lentiviral vector of interest by using a second generation lentivirus packaging system.293T cells were also used for the virus titer estimation.The multiplicity of infection (MOI) of 661W cells was detected by fluorescence microscopy.A stable transfected cell line was screened by puromycin.Immunofluorescence was used to detect transfection efficiency and cytochrome C oxidase Ⅳ(COXⅣ)-OGG1 co-localization.OGG1 mRNA and protein expression levels were detected by real-time qantitative PCR (QPCR) and Western blot.

Results

Sequencing results showed that the inserted sequence in the over-expression plasmid was consistent with the mouse OGG1 (NM_010957.4) gene sequence in the gene library.The original lentiviral titer after packaging and purification was between 2.0×107 to 6.0×107 TU/ml.The optimal MOI of 661W cells was 40, and puromycin with a concentration of 4.0 μg/ml successfully screened stable transformation.The transfection efficiency was up to 100% after screening.Immunofluorescence demonstrated successful co-localization of OGG1 and COXⅣ.The relative expression levels of OGG1 mRNA in the blank control group, OGG1 group, overexpression control group, shRNA group and low expression control group were 1.000±0.000, 41.581±12.206, 0.888±0.056, 0.239±0.121 and 1.081±0.083, and the relative expression levels of OGG1 protein were 1.029±0.153, 1.657±0.237, 0.752±0.143, 0.471±0.149 and 1.036±0.185, respectively, with significant differences between them (F=44.654, 30.948; both at P<0.05), the relative expression levels of OGG1 mRNA and protein in the OGG1 group were significantly higher than those in the overexpression control group, the relative expression levels of OGG1 mRNA and protein in the shRNA group were significantly lower than those in the lower expression control group, with significant differences between them (all at P<0.05).

Conclusions

The mito-OGG1 overexpression and OGG1 knockdown models of 661W cells are successfully constructed, which provides the preliminary experimental basis for follow-up study.

Key words: Lentivirus vector; Overexpression; Short hairpin RNA; OGG1 gene; 661W cell; Mitochondrial targeting sequence

Contributor Information

Li Han
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Guangzhou 510060, China
Ma Wei
Shi Kangpei
Wang Lei
Huang Hao
Wu Wenbin
Zhang Xiaotong
Zhu Xiaobo
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Updated: October 16, 2019 — 11:28 am