Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, and UPMC Hillman Cancer Center, Pittsburgh, PA, USA.
Department of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA, USA.
Methods Mol Biol. 2022;2444:141-159. doi: 10.1007/978-1-0716-2063-2_9.
Mammalian telomeres are guanine-rich sequences which cap the ends of linear chromosomes. While recognized as sites sensitive to oxidative stress, studies on the consequences of oxidative damage to telomeres have been primarily limited to experimental conditions which cause oxidative damage throughout the whole genome and cell. We developed a chemoptogenetic tool (FAP-mCER-TRF1) to specifically induce singlet oxygen at telomeres, resulting in the formation of the common oxidative lesion 8-oxo-guanine. Here, we describe this tool and detail how to generate cell lines which express FAP-mCER-TRF1 at telomeres and verify the formation of 8-oxo-guanine.
哺乳动物端粒是富含鸟嘌呤的序列,它们覆盖在线性染色体的末端。虽然已经认识到端粒是对氧化应激敏感的部位,但对端粒氧化损伤后果的研究主要局限于在整个基因组和细胞中引起氧化损伤的实验条件。我们开发了一种化学遗传学工具(FAP-mCER-TRF1),专门在端粒处诱导单线态氧,导致常见的氧化损伤 8-氧鸟嘌呤的形成。在这里,我们描述了这个工具,并详细介绍了如何生成在端粒处表达 FAP-mCER-TRF1 的细胞系,并验证 8-氧鸟嘌呤的形成。