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首页> 美国卫生研究院文献>Wiley-Blackwell Online Open >The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes Part I: Isolation structural genetic and biochemical characterization
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The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes Part I: Isolation structural genetic and biochemical characterization

机译：基于MutaMouse原代肝细胞的体外致突变性测定方法的开发和预验证第一部分：分离结构遗传和生化特性

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摘要

To develop an improved in vitro mammalian cell gene mutation assay, it is imperative to address the known deficiencies associated with existing assays. Primary hepatocytes isolated from the MutaMouse are ideal for an in vitro gene mutation assay due to their metabolic competence, their “normal” karyotype (i.e., neither transformed nor immortalized), and the presence of the MutaMouse transgene for rapid and reliable mutation scoring. The cells were extensively characterized to confirm their utility. Freshly isolated cells were found to have a hepatocyte‐like morphology, predominantly consisting of binucleated cells. These cells maintain hepatocyte‐specific markers for up to 3 days in culture. Analyses revealed a normal murine hepatocyte karyotype with a modal ploidy number of 4n. Fluorescence in situ hybridization analysis confirmed the presence of the lambda shuttle vector on chromosome 3. The doubling time was determined to be 22.5 ± 3.3 h. Gene expression and enzymatic activity of key Phase I and Phase II metabolic enzymes were maintained for at least 8 and 24 h in culture, respectively. Exposure to β‐naphthoflavone led to approximately 900‐ and 9‐fold increases in Cyp1a1 and Cyp1a2 gene expression, respectively, and approximately twofold induction in cytochrome P450 (CYP) 1A1/1A2 activity. Exposure to phenobarbital resulted in an approximately twofold increase in CYP 2B6 enzyme activity. Following this characterization, it is evident that MutaMouse primary hepatocytes have considerable promise for in vitro mutagenicity assessment. The performance of these cells in an in vitro gene mutation assay is assessed in Part II. Environ. Mol. Mutagen. 60:331–347, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.
机译：为了开发改进的体外哺乳动物细胞基因突变测定法，必须解决与现有测定法相关的已知缺陷。从MutaMouse分离的原代肝细胞具有代谢能力，“正常”核型（即既未转化也不会永生化）以及MutaMouse转基因的存在，可快速，可靠地进行突变评分，因此非常适合用于体外基因突变测定。对细胞进行了广泛表征，以确认其实用性。发现新鲜分离的细胞具有肝细胞样形态，主要由双核细胞组成。这些细胞在培养中最多可维持肝细胞特异性标记达3天。分析显示正常小鼠肝细胞核型，其模态倍数为4n。荧光原位杂交分析证实了λ穿梭载体在3号染色体上的存在。倍增时间确定为22.5±3.3 h。关键的I期和II期代谢酶的基因表达和酶活性分别在培养物中维持至少8小时和24小时。暴露于β-萘黄酮会分别导致Cyp1a1和Cyp1a2基因表达分别增加900和9倍，并诱导细胞色素P450（CYP）1A1 / 1A2活性增加约2倍。暴露于苯巴比妥导致CYP 2B6酶活性增加约两倍。遵循此特征，很明显MutaMouse原代肝细胞对于体外诱变性评估具有相当大的希望。在第二部分中评估了这些细胞在体外基因突变测定中的性能。环境。大声笑诱变剂。 60：331–347，2019.©2018作者。 Wiley Periodicals，Inc.代表环境诱变学会出版的《环境和分子诱变》。

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