GENE TARGETING OF PROTEIN TYROSINE PHOSPHATASE PTP-J

蛋白质酪氨酸磷酸酶 PTP-J 的基因靶向

• 批准号: 10670305
• 负责人: KISHIHARA Kenji
• 金额: $ 2.05万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670305/
• 关键词: Protein tyrosin phosphatase; Gene targeing; Gene expression

To elucidate phisiological roles of a protein tyrosine phosphatase, PTP-J, PTP-J-deficient (PTP-JィイD1-/-ィエD1 mice were made using gene targeting technology. Based on an approximately 8.7kb genomic DAN fragment containing a part of the PTP-J gene cloned by screening of a 129/J mouse-derived genomic DNA library, a recombinant plasmid vector was constructed. The linearized plasmid vector DNA was transfected into embryonic stem (ES) cells by electroporation, followed by culturing them in the presence of G418. ES cells with an expected mutation by homologous recombination were obtained at a frequency of 1/200. Finally, PTP-J-deficient mice were generated by microinjection of the ES cells into C57BL/6 mouse-derived blastocysts and repetitive mating of the resultant chimeric mice and agouti mice with C57BL/6 mice. No significant abnormalities were observed in PTP-JィイD1-/-ィエD1 mice in respect of development and reproduction. Moreover, flow cytometric analysis of immunocompetent cells showed normal size and proportion of them, suggesting that PTP-J is not essentil for development and differentiation of immune cells. At present, the tyrosine phosphorylation state of cytosolic proteins in PTP-J -expressing cells is in analysis to find a substrate of PTP-J.To examine the regulation mechanism of PTP-J expression in T-lymphoma cell lines, Jurkat and MOLT-4, effects of various inhibitors and simulators of signaling molecules on the PTP-J expression in them were analyzed. As the results, PKC, PTPs, CaィイD12+ィエD1-calmodulin-dependent protein kinase IV and Ras were involved in the regulation of the PTP-J transcription.

为了阐明蛋白酪氨酸磷酸酶PTP-J的生理学作用，使用基因打靶技术制备PTP-J缺陷型（PTP-J缺陷型）D1-/-PTP-J缺陷型D1小鼠。从129/J小鼠基因组DNA文库中筛选到约8.7kb的含有PTP-J基因部分序列的DNA片段，构建了重组质粒载体。通过电穿孔将线性化质粒载体DNA转染到胚胎干（ES）细胞中，随后在G418存在下培养它们。通过同源重组以1/200的频率获得具有预期突变的ES细胞。最后，通过将ES细胞显微注射到C57 BL/6小鼠来源的胚泡中，并将所得嵌合小鼠和无孕小鼠与C57 BL/6小鼠重复交配，产生PTP-J缺陷型小鼠。在PTP-J β D1-/-β D1小鼠中未观察到发育和生殖方面的显著异常。免疫活性细胞的流式细胞仪分析显示其大小和比例正常，提示PTP-J对免疫细胞的发育和分化不是必需的。目前，PTP-J表达细胞中胞浆蛋白的酪氨酸磷酸化状态正在分析中，以寻找PTP-J的底物。为了研究T淋巴瘤细胞系Jurkat和MOLT-4中PTP-J表达的调控机制，分析了各种信号分子的抑制剂和模拟剂对它们中PTP-J表达的影响。结果表明，PKC、PTPs、Ca ~（2+）D_12 + Ca ~（2+）D_1-CaM依赖性蛋白激酶IV和Ras参与了PTP-J转录的调控。