Abstract

Transcription factor NRF2 is involved in inflammatory reactions, maintenance of redox balance, metabolism of xenobiotics, and is of particular interest for studying aging. In the present work, the CRISPR/Cas9 genome editing technology was used to generate the NRF2^ΔNeh2 mice containing a substitution of eight amino acid residues at the N-terminus of the NRF2 protein, upstream of the functional Neh2 domain, which ensures binding of NRF2 to its inhibitor KEAP1. Heterozygote NRF2^wt/ΔNeh2 mice gave birth to homozygous mice with lower than expected frequency, accompanied by their increased embryonic lethality and visual signs of anemia. Mouse embryonic fibroblasts (MEFs) from the NRF2^{ΔNeh2/ΔNeh2} homozygotes showed impaired resistance to oxidative stress compared to the wild-type MEFs. The tissues of homozygous NRF2^{ΔNeh2/ΔNeh2} animals had a decreased expression of the NRF2 target genes: NAD(P)H:Quinone oxidoreductase-1 (Nqo1); aldehyde oxidase-1 (Aox1); glutathione-S-transferase A4 (Gsta4); while relative mRNA levels of the monocyte chemoattractant protein 1 (Ccl2), vascular cell adhesion molecule 1 (Vcam1), and chemokine Cxcl8 was increased. Thus, the resulting mutation in the Nfe2l2 gene coding for NRF2, partially impaired function of this transcription factor, expanding our insights into the functional role of the unstructured N-terminus of NRF2. The obtained NRF2^ΔNeh2 mouse line can be used as a model object for studying various pathologies associated with oxidative stress and inflammation.

中文翻译：

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NFE2L2 (NRF2) 基因突变的新小鼠品系

摘要

转录因子 NRF2 参与炎症反应、氧化还原平衡的维持、外源物质的代谢，并且对于研究衰老特别感兴趣。在本工作中，利用CRISPR/Cas9基因组编辑技术生成了NRF2 ^ΔNeh2小鼠，该小鼠在NRF2蛋白的N端、功能性Neh2结构域的上游含有8个氨基酸残基的取代，这确保了NRF2的结合其抑制剂 KEAP1。杂合子 NRF2 ^wt/ΔNeh2小鼠产下纯合子小鼠的频率低于预期，并伴有胚胎致死率增加和贫血的视觉症状。与野生型 MEF 相比，来自 NRF2 ^{ΔNeh2/ΔNeh2纯合子的小鼠胚胎成纤维细胞 (MEF) 显示出对氧化应激的抵抗力受损。纯合 NRF2 ΔNeh2/ΔNeh2}动物的组织中NRF2 靶基因 NAD(P)H:Quinone oxyreductase-1 ( Nqo1 )的表达降低；醛氧化酶-1 ( Aox1 ); 谷胱甘肽-S-转移酶 A4 ( Gsta4 )；而单核细胞趋化蛋白1（Ccl2）、血管细胞粘附分子1（Vcam1）和趋化因子Cxcl8的相对mRNA水平增加。因此，编码 NRF2 的Nfe2l2基因发生突变，该转录因子的功能部分受损，从而扩大了我们对 NRF2 非结构化 N 末端功能作用的认识。获得的NRF2 ^ΔNeh2小鼠系可以作为研究与氧化应激和炎症相关的各种病理学的模型对象。