Gene targeting of plasminogen activator inhibitor 2

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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.