Establishment of Neh2-Cre:tdTomato reporter mouse for monitoring the exposure history to electrophilic stress
Establishment of Neh2-Cre:tdTomato reporter mouse for monitoring the exposure history to electrophilic stress
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Neh2-Cre:tdTomato 报告小鼠的建立，用于监测亲电应激暴露史
DOI：
10.1016/j.freeradbiomed.2022.11.004
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Hozumi Motohashi
中科院分区：
文献类型：
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作者：
Hiroshi Kitamura;Tetsuya Oishi;Shohei Murakami;Tomoe Yamada-Kato;Isao Okunishi;Masayuki Yamamoto;Yukio Katori;Hozumi Motohashi
细胞经常暴露在外源性和内源性氧化还原干扰中，并在刺激下发挥其保护机制。Keap1-NRF2系统在对抗氧化损伤中起着关键作用。由于NRF2激活的暂时性，识别NRF2在系统刺激下被激活的细胞有时并不容易。为了在单细胞分辨率下检测亲电应激反应，我们的目标是在这项研究中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跟踪策略来稳定检测报告基因的表达，而不是实时监测细胞反应。我们建立了表达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，作为降解物并介导Keap1依赖的降解和电泳法诱导的稳定，Neh2-Cre有望在亲电性物质的响应下被激活。将Neh2-Cre转基因小鼠与rosa26-loxP-Stop-loxP-tdTomato报告小鼠(ROSA-LSL-tdTomato小鼠)杂交。复合突变报告小鼠在反复给予NRF2诱导的电泳体之一CDDO-Im后，在不同的器官中显示出tdTomato阳性细胞的积累。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。
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.