A Knockout/Knockin Approach to Evaluate the Function of ARNT and ARNT2 Protein

评估 ARNT 和 ARNT2 蛋白功能的敲除/敲入方法

• 批准号: 7878404
• 负责人: MICHAEL J KERN
• 金额: $ 19.45万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7878404
• 关键词: ARNT protein; ARNT2 gene; Accounting; Address; Amino Acids; Animals; Aryl Hydrocarbon Receptor; Biochemistry; Blood Vessels; Cell Line; Chemicals; Conflict (Psychology); DNA; Data; Defect; Development; Diabetes Mellitus; Dimerization; Down Syndrome; Elements; Embryo; Exhibits; Financial compensation; Future; Gene Targeting; Generations; Genes; Goals; Grant; Helix-Turn-Helix Motifs; Hormones; Hypoxia Inducible Factor; In Vitro; Invertebrates; Knock-out; Literature; Malignant Neoplasms; Mammals; Mediating; Mission; Modeling; Modification; Mus; Non-Insulin-Dependent Diabetes Mellitus; Open Reading Frames; Oxygen; Pancreas; Pathology; Pathway interactions; Pattern; Perinatal; Pharmacology and Toxicology; Phenotype; Physiological; Protein Family; Proteins; Reagent; Regulatory Element; Signal Pathway; Signal Transduction; Testing; Tissues; Transgenic Mice; Transgenic Organisms; United States National Institutes of Health; body system; human ARNT protein; in vivo; member; mouse model; nervous system disorder; novel; protein expression; protein function; public health relevance; receptor; response; single-minded protein; transcription factor

DESCRIPTION (provided by applicant): The expression of the aryl hydrocarbon nuclear translocator (ARNT and ARNT2) proteins are essential for development of multiple organ systems in mammals and are required for physiological pathways that respond to important exogenous and endogenous environmental signals (chemicals, hormones, oxygen). Reductions in ARNT protein expression in the pancreas have been implicated in type 2 diabetes, while elevated expression of its bHLH/PAS dimerization partner SIM2, has been implicated in Downs Syndrome. ARNT and ARNT2 proteins exhibit 80-90% amino acid identity in the bHLH and PAS domains. There is conflicting data in the literature regarding the potential for these two proteins to perform the same function in vitro and other conflicting data concerning the pattern of expression of ARNT2 in vivo and in cell lines. Given the importance of these proteins in many of the same signaling pathways, it is intriguing that targeted disruption of the Arnt gene in mice results in embryonic lethality due to vascular defects, while the targeted disruption of the Arnt2 gene is characterized by perinatal lethality involving CNS deficits. Despite the overlaps in tissue expression and the high level of amino acid identity between these two proteins, the prevailing interpretation of the transgenic results has resulted in the hypothesis that ARNT and ARNT2 proteins are not capable of compensating for the loss of the other protein in vivo. However, there are several explanations that may account for these findings such as i) each ARNT has a distinct level of protein expression in a given tissue that does not support a specific pathway, ii) tissue specific modifications of the different ARNTs impact their function, or iii) each ARNT has one or more distinct functions not shared by the other. A powerful approach to begin to address these important questions is to utilize a knockin/knockout strategy whereby the ARNT2 ORF is inserted into the Arnt locus while simultaneously abolishing ARNT protein expression. In this approach, ARNT2 expression will be controlled by all the regulatory elements normally reserved for ARNT and result in the physiological expression of ARNT2 in a mouse that does not express ARNT. The generation of this novel gene-targeted model will allow a direct test of the hypothesis that the concentration of ARNT2 protein expression in tissues contribute to the lack of compensation in Arnt-/- animals. The integrated set of aims will show how the level of expression of both ARNT and ARNT2 protein impact established physiological pathways, the response to important exogenous and endogenous environmental signals and development. Successful completion will illustrate the utility of such a transgenic approach to assess the function of similar proteins, but also may produce key models for the analysis of development (via SIM and HIF proteins), pharmacology and toxicology (via HIF and AHR proteins), diabetes (via reductions in ARNT) and cancer (via AHR and HIF proteins). PUBLIC HEALTH RELEVANCE: The expression of the aryl hydrocarbon nuclear translocator (ARNT and ARNT2) proteins are essential for development of multiple organ systems in mouse and are required for established physiological pathways that respond to important exogenous and endogenous environmental signals (chemicals, hormones, oxygen). ARNT is a key protein in development of cancer and perturbation of ARNT and ARNT2 expression has been associated with diabetes and neurological disorders.

描述（由申请人提供）：芳烃核易位蛋白（ARNT 和 ARNT2）的表达对于哺乳动物多器官系统的发育至关重要，并且是响应重要的外源和内源环境信号（化学物质、激素）的生理途径所必需的。 、氧气）。胰腺中 ARNT 蛋白表达的减少与 2 型糖尿病有关，而其 bHLH/PAS 二聚化伙伴 SIM2 的表达升高与唐氏综合症有关。 ARNT 和 ARNT2 蛋白在 bHLH 和 PAS 结构域中表现出 80-90% 的氨基酸同一性。文献中存在关于这两种蛋白质在体外执行相同功能的潜力的相互矛盾的数据，以及关于 ARNT2 在体内和细胞系中的表达模式的其他相互矛盾的数据。鉴于这些蛋白质在许多相同信号通路中的重要性，有趣的是，小鼠中 Arnt 基因的靶向破坏会因血管缺陷而导致胚胎致死，而 Arnt2 基因的靶向破坏则导致涉及中枢神经系统的围产期致死。赤字。尽管这两种蛋白质之间存在组织表达重叠和高水平的氨基酸同一性，但对转基因结果的普遍解释导致了这样的假设：ARNT 和 ARNT2 蛋白质无法补偿体内另一种蛋白质的损失。 。然而，有几种解释可以解释这些发现，例如 i) 每个 ARNT 在不支持特定途径的给定组织中具有不同的蛋白质表达水平，ii) 不同 ARNT 的组织特异性修饰影响其功能，或 iii) 每个 ARNT 具有一个或多个彼此不共享的不同功能。解决这些重要问题的一个有效方法是利用敲入/敲除策略，将 ARNT2 ORF 插入 Arnt 基因座，同时消除 ARNT 蛋白表达。在这种方法中，ARNT2 表达将受到通常为 ARNT 保留的所有调控元件的控制，并导致 ARNT2 在不表达 ARNT 的小鼠中生理表达。这种新型基因靶向模型的产生将允许直接检验以下假设：组织中 ARNT2 蛋白表达的浓度导致 Arnt-/- 动物补偿的缺乏。综合目标将展示 ARNT 和 ARNT2 蛋白的表达水平如何影响已建立的生理途径、对重要外源和内源环境信号的反应以及发育。成功完成将说明这种转基因方法在评估类似蛋白质功能方面的实用性，而且还可能产生用于分析发育（通过 SIM 和 HIF 蛋白质）、药理学和毒理学（通过 HIF 和 AHR 蛋白质）、糖尿病的关键模型（通过减少 ARNT）和癌症（通过 AHR 和 HIF 蛋白）。 公共卫生相关性：芳烃核易位蛋白（ARNT 和 ARNT2）蛋白的表达对于小鼠多器官系统的发育至关重要，并且是建立响应重要外源和内源环境信号（化学物质、激素、氧气）的生理途径所必需的。 ）。 ARNT 是癌症发展中的关键蛋白，ARNT 和 ARNT2 表达的扰动与糖尿病和神经系统疾病有关。