Hepatocyte-targeted somatic-cell genetic complementation in mice

小鼠肝细胞靶向体细胞遗传互补

• 批准号: 10017365
• 负责人: EDWARD E SCHMIDT
• 金额: $ 18万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017365
• 关键词: Acetaminophen; Active Sites; Aging; Animals; Antioxidants; Arsenic; Biocompatible Materials; CRISPR screen; CRISPR/Cas technology; Candidate Disease Gene; Cells; Cisplatin; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Consumption; Cultured Cells; Development; Disease; Disulfides; Enzymes; Exposure to; Family member; Genes; Genetic; Genetic Models; Glutathione; Glutathione Reductase; Health; Hepatic; Hepatocyte; Homeostasis; In Situ; Inflammatory; Knock-out; Liver; Logistics; Malignant Neoplasms; Mammalian Cell; Mammals; Masks; Mediating; Messenger RNA; Metabolic; Metals; Methionine; Microbe; Minerals; Modeling; Molecular; Mus; N-acetyl-4-benzoquinoneimine; NADP; Nerve Degeneration; Null Lymphocytes; Nutrient; Organic Chemicals; Oxidation-Reduction; Oxidative Stress; Oxidoreductase; Pathway interactions; Patients; Peroxidases; Pharmaceutical Preparations; Plants; Process; Reperfusion Therapy; Research; Resistance; Ribonucleotide Reductase; Role; Somatic Cell Genetics; Source; Sulfur; Supporting Cell; System; TXN gene; Testing; Therapeutic; Toxic Environmental Substances; Toxicant exposure; Toxin; Uncertainty; animal model development; base; cell type; electron donor; genome editing; genome wide screen; glutaredoxin; improved; innovation; liver ischemia; methionine sulfoxide reductase; microbial; mouse model; novel; novel strategies; peroxiredoxin; prevent; redoxin; stem cells; thioredoxin reductase 1; transcriptome; vector-borne; whole genome

Summary/Abstract Disulfide reductase-driven antioxidant defenses prevent molecular damage that can contribute to inflammatory diseases, neurodegeneration, stem cell depletion, aging, and cancers. NADPH, generated from NADP+ using energetic nutrients, is the electron-donor for most biosynthetic, homeostatic, and cytoprotective reductions, but only two enzymes can use NADPH to reduce cytosolic disulfides: thioredoxin reductase-1 (TrxR1) and glutathione reductase (Gsr). Electrophilic toxins can coincidentally inhibit both the TrxR1 and Gsr pathways in liver. These include environmental metal/metalloids (e.g., arsenic), drugs (e.g., cisplatin) or drug metabolites (e.g., NAPQI from acetaminophen), and many organic toxins from plants or microbes. Mice with liver-specific disruptions of both TrxR1+Gsr (TrxR1/Gsr-null), which provide a useful genetic model for such situations, have revealed surprising robustness in the disulfide reductase systems, including a previously unrecognized methionine (Met)-fueled NADPH-independent system that sustains redox homeostasis in TrxR1/Gsr-null livers. These models reveal that mammals, unlike microbes, have unexpected sources and distribution- mechanisms to supply disulfide reducing power when the canonical pathways are compromised. We hypothesize that realigned metabolic activities and expanded functionality of Trx- and glutaredoxin (Grx)-family members provides support for essential reductase activities, when needed. A better understanding of these systems promises to provide improved therapeutic avenues for rescuing liver- and patient-health following severe oxidative stress or toxic exposures. Testing this hypothesis, however, will require development of innovative approaches to detect the putative complementary activities. Here we propose two specific aims that will use a novel CRISPR/Cas9 gene disruption approach in genetically modified mouse livers to (i) define the respective roles of Grx family members in distributing reducing power when Trx1 is disrupted and (ii) perform an innovative screen to identify genes supporting redox homeostasis upon co-disruption of TrxR1 and Gsr. Synopsis: This project will use innovative approaches for genome-editing-enhanced somatic cell genetic complementation in mouse liver to better define the pathways that support disulfide reductase systems when the canonical pathways become compromised. Consistent with PA-16-141: “Development of animal models and related biological materials for research (R21)”, this project develops a new approach for performing genetic complementation studies in mouse hepatocytes in situ.

摘要/摘要 二硫化物还原酶驱动的抗氧化剂防御系统可防止导致炎症的分子损伤 疾病、神经退化、干细胞枯竭、衰老和癌症。NADPH，使用NADP+生成 高能营养素是大多数生物合成、动态平衡和细胞保护还原的电子供体， 但只有两种酶可以利用NADPH还原胞液中的二硫化物：硫氧还蛋白还原酶-1(TrxR1)和 谷胱甘肽还原酶(GSR)。亲电毒素可同时抑制TrxR1和GSR信号通路 肝脏。这些包括环境金属/类金属(如砷)、药物(如顺铂)或药物代谢物。 (例如，扑热息痛的NAPQI)，以及来自植物或微生物的许多有机毒素。肝脏特异的小鼠 TrxR1+GSR(TrxR1/GSR-空)的中断为这种情况提供了有用的遗传模型，具有 在二硫化物还原酶系统中发现了令人惊讶的健壮性，包括以前未被识别的 蛋氨酸(Met)驱动的NADPH非依赖性系统，在TrxR1/GSR阴性肝脏中维持氧化还原动态平衡。 这些模型表明，哺乳动物与微生物不同，它们有意想不到的来源和分布-- 当正则途径受损时，提供二硫化物还原动力的机制。我们 重新调整Trx-和GRX家族的代谢活动和扩展功能的假设 成员在需要时为基本的还原酶活动提供支持。更好地理解这些 系统承诺为挽救肝脏和患者健康提供更好的治疗途径 严重的氧化应激或中毒暴露。然而，测试这一假设将需要开发 发现假定的补充活动的创新方法。 在这里，我们提出了两个具体的目标，将使用一种新的CRISPR/Cas9基因破坏方法在遗传学上 修饰小鼠肝脏以(I)定义GRX家族成员在分配还原能力中的各自角色 当Trx1被破坏时以及(Ii)进行创新筛选以识别支持氧化还原动态平衡的基因 当TrxR1和GSR共同中断时。 简介：该项目将使用基因组编辑增强体细胞遗传学的创新方法 在小鼠肝脏中的互补作用，以更好地确定支持二硫化物还原酶系统的途径 规范路径变得不堪一击。与PA-16-141一致：“动物模型的发展 和相关生物材料用于研究(R21)，该项目开发了一种新的方法来执行 小鼠肝细胞遗传互补的原位研究。