GENE TARGETING OF PROTEIN TYROSINE PHOSPHATASE PTP-J

To elucidate phisiological roles of a protein tyrosine phosphatase， PTP-J，PTP-J-deficient（PTP-JイD1-/-イエD1 mice were made using gene targeting technology）。基于一个接近8.7 kb的基因组DAN片段，包含PTP-J基因的一部分，通过筛选的129/J小鼠衍生基因组DNA库，一个重组的质粒载体被构建。线性的等离子体载体DNA被转染到胚胎干（ES）细胞中，由电泳在G418的存在中跟随。ES细胞与一个预期的突变由同源重组发生在1/200的频率。最后，PTP-J-deficient mice由ES细胞的微注射产生C57 BL/6小鼠衍生出的blastocystem和与C57 BL/6小鼠的重复匹配。在PTP-J中没有significant abManagement alities were observed in PTP-J中的D1-/-D1 mice in respect of Oracle and reproduction。Moreover，对免疫细胞的流动细胞分析显示了正常大小和它们的比例，建议PTP-J不是免疫细胞的开发和差异的基本要素。目前，PTP-J-表达细胞中细胞质蛋白的酪氨酸磷酸化状态在分析中找到PTP-J的底物，以测试T-淋巴瘤细胞线、Jurkat和MOLT-4中PTP-J表达的调节机制，对各种抑制剂的影响和PTP-J表达中的信号分子的模拟器。根据结果， PKC， PTPs，Ca-D12 +-钙调蛋白依赖蛋白激酶IV和Ras被纳入PTP-J转录的规定。

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.