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Vol 55(2021) N 5 p. 742-751; DOI 10.1134/S0026893321040099 Full Text

X.X. Wang1*, H.J. Jia2, Y.R. Lv1,2, H.H. Sun1,2, X.L. Wei3, J.Y. Tan3, Z.Z. Jing2**

A Luciferase-EGFP Reporter System for the Evaluation of DNA Methylation in Mammalian Cells

1School of Public Health, Lanzhou University, Lanzhou, 730000 China
2State Key Laboratory of Veterinary of Etiological Biology, Key Laboratory of Veterinary Public Health of Agricultural Ministry, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730000 China
3Institute of Immunology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, 730000 China

*wangxiaoxia@lzu.edu.cn
**jingzhizhong@caas.cn
Received - 2020-06-06; Revised - 2020-10-22; Accepted - 2020-11-03

DNA methylation is an essential epigenetic modification involved in numerous biological processes. Here, we present a cell-based system pLTR-Luc2P-EGFP for evaluation of DNA methylation in mammalian cells. In this system, the expression of reporter gene luciferase2P (Luc2P)-EGFP is under the control of HIV-1 promoter 5' long terminal repeat (LTR), which contains multiple CpG sites. Once these sites are methylated, the expression of Luc2P-EGFP is turned off, which may be visualized under fluorescence microscopy, with quantification performed in luciferase activity assay. As a proof of principle, pLTR-Luc2P-EGFP was methylated in vitro, and transfected into 293T cells, where the reduction of Luc2P-EGFP expression was confirmed. Premixed reporter DNA samples with the methylation levels varying from 0 to 100% were used for quantitative measurements of DNA methylation. The resulting standard curves indicated the accuracy of luciferase activity exceeding that of the Western blotting against EGFP. The Bland-Altman analysis showed that data from luciferase activity assay were in good agreement with the actual DNA methylation levels. In summary, we have established a reporter system coupled with reliable detection technique capable of efficient quantifying the changes in methylation in mammalian cells. This system may be utilized as a high throughput screening tool for identifying molecules that modulate DNA methylation.

Luc2P firefly luciferase, cell-based reporter system, HIV-1 promoter 5' LTR, DNA methylation



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