Abstract

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.

中文翻译：

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用于评估哺乳动物细胞 DNA 甲基化的荧光素酶-EGFP 报告系统

摘要

DNA甲基化是一种重要的表观遗传修饰，涉及许多生物学过程。在这里，我们提出了一种基于细胞的系统 pLTR-Luc2P-EGFP，用于评估哺乳动物细胞中的 DNA 甲基化。在该系统中，报告基因 luciferase2P (Luc2P)-EGFP 的表达受 HIV-1 启动子 5' 长末端重复 (LTR) 的控制，其中包含多个 CpG 位点。一旦这些位点被甲基化，Luc2P-EGFP 的表达就会关闭，这可以在荧光显微镜下观察到，并在荧光素酶活性测定中进行量化。作为原理证明，pLTR-Luc2P-EGFP 在体外被甲基化，并转染到 293T 细胞中，证实了 Luc2P-EGFP 表达的降低。甲基化水平从 0% 到 100% 不等的预混合报告 DNA 样品用于 DNA 甲基化的定量测量。得到的标准曲线表明荧光素酶活性的准确性超过了针对 EGFP 的蛋白质印迹的准确性。Bland-Altman 分析显示来自荧光素酶活性测定的数据与实际 DNA 甲基化水平非常一致。总之，我们建立了一个报告系统，结合可靠的检测技术，能够有效量化哺乳动物细胞中甲基化的变化。该系统可用作高通量筛选工具，用于鉴定调节 DNA 甲基化的分子。Bland-Altman 分析显示来自荧光素酶活性测定的数据与实际 DNA 甲基化水平非常吻合。总之，我们建立了一个报告系统，结合可靠的检测技术，能够有效量化哺乳动物细胞中甲基化的变化。该系统可用作高通量筛选工具，用于鉴定调节 DNA 甲基化的分子。Bland-Altman 分析显示来自荧光素酶活性测定的数据与实际 DNA 甲基化水平非常吻合。总之，我们建立了一个报告系统，结合可靠的检测技术，能够有效量化哺乳动物细胞中甲基化的变化。该系统可用作高通量筛选工具，用于鉴定调节 DNA 甲基化的分子。