GENE TARGETING OF PROTEIN TYROSINE PHOSPHATASE PTP-J

To elucidate phisiological roles of a protein tyrosine phosphatase，PTP-J-deficient(PTP-J I D1-/-I 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 a129/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/6mouse-derived blastocysts and repetitive mating of the resultant chimeric mice and agouti mice with C57BL/6mice.No significant abnormalities were observed in PTP-J I D1-/-I 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 I D12+ii D1-calmodulin-dependent protein kinase IV and Ras were involved in the regulation of the PTP-J transcription.

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.