Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aobaku, Sendai, 980-8575, Japan.
Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aobaku, Sendai, 980-8575, Japan; Department of Otolaryngology-Head and Neck Surgery, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aobaku, Sendai, 980-8574, Japan.
Free Radic Biol Med. 2022 Nov 20;193(Pt 2):610-619. doi: 10.1016/j.freeradbiomed.2022.11.004. Epub 2022 Nov 9.
Cells are often exposed to exogenous and endogenous redox disturbances and exert their protective mechanisms in response to stimuli. The KEAP1-NRF2 system plays pivotal roles in counteracting oxidative damage. Due to the transient nature of NRF2 activation, the identification of cells in which NRF2 is activated in response to systemic stimuli is sometimes not easy. To examine the electrophilic stress response at a single-cell resolution, we aimed to develop a new reporter mouse in this study. A cell-tracing strategy exploiting Cre recombinase-mediated activation of a reporter gene was chosen for stable detection of reporter expression instead of real-time monitoring of the cellular response. We established a transgenic mouse line expressing the Neh2-Cre recombinase fusion protein. As Neh2 is an amino-terminal domain of NRF2 that serves as a degron and mediates KEAP1-dependent degradation and electrophile-inducible stabilization, Neh2-Cre was expected to be activated in response to electrophiles. The Neh2-Cre transgenic mouse was crossed with the ROSA26-loxP-stop-loxP-tdTomato reporter mouse (ROSA-LSL-tdTomato mouse). The compound mutant reporter mice exhibited accumulation of tdTomato-positive cells in various organs after repeated administration of CDDO-Im, one of the NRF2-inducing electrophiles. The mice were also successfully used for the detection of cells that experienced a cisplatin-induced electrophilic stress response.
细胞经常受到外源性和内源性氧化还原紊乱的影响，并通过其保护机制对刺激做出反应。KEAP1-NRF2 系统在对抗氧化损伤方面起着关键作用。由于 NRF2 激活的瞬时性，有时不容易确定对系统刺激做出 NRF2 激活反应的细胞。为了在单细胞分辨率下检查亲电应激反应，本研究旨在开发一种新的报告小鼠。选择了一种利用 Cre 重组酶介导的报告基因激活的细胞追踪策略，用于稳定检测报告表达，而不是实时监测细胞反应。我们建立了一种表达 Neh2-Cre 重组酶融合蛋白的转基因小鼠系。由于 Neh2 是 NRF2 的氨基末端结构域，作为一种降解物，介导 KEAP1 依赖性降解和亲电诱导的稳定化，因此预计 Neh2-Cre 会对亲电物质做出反应。Neh2-Cre 转基因小鼠与 ROSA26-loxP-stop-loxP-tdTomato 报告鼠（ROSA-LSL-tdTomato 小鼠）杂交。在重复给予 NRF2 诱导亲电物 CDDO-Im 后，复合突变体报告小鼠的各种器官中积累了 tdTomato 阳性细胞。这些小鼠还成功地用于检测经历顺铂诱导的亲电应激反应的细胞。
