APPLICATION OF GENE MANIPULATION TECHNOLOGY TO GENERATION OF MODEL ANIMALS FOR ENVIRONMENTAL ASSESSMENT

基因操作技术在环境评估模型动物生成中的应用

• 批准号: 12794009
• 负责人: YAMAMOTO Masayuki
• 金额: $ 15.36万
• 依托单位: UNIVERSITY OF TSUKUBA
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for University and Society Collaboration
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12794009/
• 关键词: DETOXIFICATION; XENOBIOTICS; TRANSCRIPTIONAL REGULATION; MODEL ANIMALS; bZip TRANSCRIPTION FACTORS; bHLH-PAS TRANSCRIPTION FACTORS; bZip型転写因子

1. Generation of AhR-overexpressing mouse. Mice carrying AhR cDNA driven by keratin14 promoter displayed inflammation and hyperkeratinization of the skin. These mice are now under examination whether they are more vulnerable to chemical carcinogens.2. Generation of human AhR (hAhR)-knock-in mice. The mouse AhR gene was replaced with human AhR cDNA by homologous recombination in ES cells. By injecting the ES cells into blastcysts, we generated hAhR-knock-in mouse expressing human AhR molecule instead of mouse AhR. When exposed to 3-methylcholanthrene, hAhR-knock-in mice displayed the comparable induction of the phase I enzyme genes with the control animals. When TCDD was used, hAhR-knock in mice showed much weaker response than the control mice. We suspect that the response observed in hAhR-knock in mouse mimics that of the humans. This mouse will serve as an animal model for the assessment of the toxicity of environmental chemicals.3. Analysis on the responses to environmental chemicals of Nrf2-deficient mice. Nrf2-deficient mice were challenged by several environmental chemicals to examine the significance of Nrf2 contribution in vivo. Nrf2-deficient mice were more sensitive to acetaminophen hepatotoxicity due to the increased level of active metabolic intermediates. Increased levels of DNA adducts was observed in Nrf2-null mutant mice after the exposure to diesel exhaust. Oral administration of benzo[a]pyrene caused severer gastric cancer in Nrf2-deficient mice than the control animals. These results indicate that Nrt2 is a key regulator of the protective responses against the toxicity of environmental chemicals.

1. AhR 过表达小鼠的生成。携带由 keratin14 启动子驱动的 AhR cDNA 的小鼠表现出皮肤炎症和过度角化。现在正在检查这些小鼠是否更容易受到化学致癌物的影响。2．人类 AhR (hAhR) 敲入小鼠的产生。在ES细胞中通过同源重组将小鼠AhR基因替换为人AhR cDNA。通过将 ES 细胞注射到囊肿中，我们产生了表达人 AhR 分子而不是小鼠 AhR 的 hAhR 敲入小鼠。当暴露于 3-甲基胆蒽时，hAhR 敲入小鼠表现出与对照动物相当的 I 相酶基因诱导。当使用 TCDD 时，hAhR 敲入小鼠的反应比对照小鼠弱得多。我们怀疑小鼠 hAhR 敲入中观察到的反应模仿了人类的反应。该小鼠将作为环境化学物质毒性评估的动物模型。 3． Nrf2缺陷小鼠对环境化学物质的反应分析。 Nrf2 缺陷小鼠受到几种环境化学物质的攻击，以检查 Nrf2 在体内贡献的重要性。由于活性代谢中间体水平增加，Nrf2 缺陷小鼠对对乙酰氨基酚的肝毒性更加敏感。在暴露于柴油机尾气后，Nrf2 缺失突变小鼠的 DNA 加合物水平增加。口服苯并[a]芘导致 Nrf2 缺陷小鼠比对照动物更严重的胃癌。这些结果表明 Nrt2 是针对环境化学品毒性的保护性反应的关键调节剂。

项目成果

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Kwak, M-K. 等人：“转录因子 Nrf2 在癌症化学预防剂诱导体内肝相 2 和抗氧化酶中的作用。3H-1, 2-二硫醇-3-硫酮”Mol。

鈴木教朗, 山本雅之: "造血幹細胞:基礎から遺伝子治療・再生医療へ 「造血幹細胞の転写調節機構」"中外医学社(印刷中).

铃木典明、山本雅之：“造血干细胞：从基础知识到基因治疗和再生医学‘造血干细胞的转录调控机制’”中外医学社（出版中）。

Cho, H.Y.et al.: "Linkage analysis of susceptibility to hyperoxic lung injury : role of Nrf2 as a candidate gene"Am. J. Res. Cell Mol. Biol.. (in press).

Cho, H.Y.等人：“高氧性肺损伤易感性的连锁分析：Nrf2 作为候选基因的作用”Am。

Cho, H.Y., Jedlika, A.E., Zhang, L.Y., Yamamoto, M., Kensler, T.W., Kleeberger, S.R.: "Linkage analysis of susceptibility to hyperoxic lung injury: role of Nrt2 as a candidate gene"Am. J. Res. Cell Mol. Biol.. 26. 175-182 (2002)

Cho, H.Y.、Jedlika, A.E.、Zhang, L.Y.、Yamamoto, M.、Kensler, T.W.、Kleeberger, S.R.：“高氧性肺损伤易感性的连锁分析：Nrt2 作为候选基因的作用”Am。

Kim, Y-C., Masutani, H., Yamaguchi, Y., Itoh, K., Yamamoto, M. and Yodoi, J.: "Hemin-induced activation of the Thioredoxin gene by Nrf2 : a differential regulation of the antioxidant responsive element (ARE) by switch of its binding factors"J. Biol. Chem.

Kim, Y-C.、Masutani, H.、Yamaguchi, Y.、Itoh, K.、Yamamoto, M. 和 Yodoi, J.：“Nrf2 血红素诱导的硫氧还蛋白基因激活：抗氧化反应元件的差异调节