Gene targeting of plasminogen activator inhibitor 2

纤溶系统不仅在血管溶栓过程中发挥重要作用，而且在伤口愈合、细胞迁移、炎症等多种生物反应中也发挥着重要作用。为了研究纤维蛋白溶解系统的调控分子之一palsminogen activator inhibitor 2 （PAI-2）的作用，我们尝试通过基因靶向培养PAI-2缺陷小鼠。我们利用含有pgk启动子驱动的新霉素耐药基因（pgk Neo）和胸苷激酶基因（TK）以及PAI-2基因DNA片段的质粒载体，制备了两种类型的质粒靶向载体pP2EX2和pP2EX8。pP2EX2和pPEX8分别用pgk Neo取代外显子II和外显子V、VI、VII以及外显子8的一部分。通过电穿孔将靶向载体导入CGR8 ES细胞，在genticin和Ganciclovir的作用下培养CGR8 ES细胞，筛选出500多个ES细胞集落建立独立克隆。经Southern blot分析，选择具有PAI-2基因重组的克隆。将重组阳性的胚胎干细胞注射到小鼠胚泡中，并转移到假妊娠小鼠体内。虽然克隆小鼠出生，但PAI-2基因重组的生发传递并不成功。因此，我们将胚胎干细胞系从CGR8改为具有生发传递能力的El4gt2a。将靶向载体pP2EX8导入El4gt2a ES细胞，选择重组阳性的ES细胞克隆。我们现在开发了三个具有PAI-2基因适当重组的胚胎干细胞克隆。

The fibrinolytic system plays an important role not only in vascular thrombolysis but also in a variety of biological reactions such as wound healing, cell migration, and inflammation. To study a role of one of the regulatory molecule of the fibrinolytic system, palsminogen activator inhibitor 2 (PAI-2), we have attempted to develop PAI-2 deficient mice by gene targeting. We made two types of plasmid targeting vector, pP2EX2 and pP2EX8, using plasmid vector containing pgk promoter-driven neomycin resistant gene (pgk Neo) and thymidine kinase gene (TK), and PAI-2 gene DNA fragments. pP2EX2 and pPEX8 were designed to replace exon II and exons V, VI, VII, and a part of exon 8 with pgk Neo respectively. Targeting vectors were introduced into CGR8 ES cells by electroporation and CGR8 ES cells were cultured in the presence of Geneticin and Ganciclovir and more than 500 ES cell colonies were selected to make independent clones. After Southern blot analysis, clones that have the recombination in the PAI-2 gene were selected. The recombination-positive ES cells were injected to mouse blast cysts and were transferred to pseudo pregnant mice. Although cimeric mice were born, germinal transmission of PAI-2 gene recombination was not successful. Thus we changed the ES cell line from CGR8 to El4gt2a which was shown to be germinal transmission competent. The targeting vector pP2EX8 was introduced into El4gt2a ES cells and recombination-positive ES cell clones were selected as before. We now developed three ES cell clones that have the appropriate recombination of the PAI-2 gene.