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Gene targeting of plasminogen activator inhibitor 2

纤溶酶原激活剂抑制剂 2 的基因靶向

基本信息

批准号：
09671132
负责人：
MIMURO Jun
金额：
$ 1.6万
依托单位：
Jichi Medical School, School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671132/
关键词：
fibrinolysis plasminogen activator inhibitor 2 gene targeting Plasminogen activator inhibitor plasminogen activator inhibitor embryonic stem cell

项目摘要

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.

纤维蛋白溶解系统不仅在血管血栓溶解中起重要作用，而且在各种生物反应如伤口愈合、细胞迁移和炎症中也起重要作用。为了研究纤溶系统的调节分子之一帕司明原激活物抑制剂2（派-2）的作用，我们尝试通过基因打靶的方法建立派-2缺陷小鼠。利用pgk启动子驱动的新霉素抗性基因（pgk Neo）、胸苷激酶基因（TK）和派-2基因DNA片段构建了两种质粒打靶载体pP 2 EX 2和pP 2 EX 8。pP 2 EX 2和pPEX 8被设计为分别用pgk Neo替换外显子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CGR 8 ES细胞，并在遗传霉素和更昔洛韦存在下培养CGR 8 ES细胞，并选择超过500个ES细胞集落以制备独立克隆。经Southern杂交分析，筛选出派-2基因重组的克隆。将重组阳性的ES细胞注射到小鼠胚泡中，并转移到假孕小鼠体内。尽管成功地产生了嵌合体小鼠，但派-2基因重组的遗传传递并不成功。因此，我们将ES细胞系从CGR 8改变为E14 gt 2a，其显示为具有生殖细胞传递能力。将靶向载体pP 2 EX 8导入E14 gt 2a ES细胞，并如前所述选择重组阳性ES细胞克隆。我们现在开发了三种具有派-2基因适当重组的ES细胞克隆。