CHARACTERIZATION AND TARGETING OF THE MURINE ALPHA(2)-ANTIPLASMIN GENE

alpha(2)-Antiplasmin (alpha(2)-AP) is the main physiological plasmin i nhibitor in mammalian plasma. As a first step toward the generation of alpha(2)-AP deficient mice the murine alpha(2)-AP gene was characteri zed and a targeting vector for homologous recombination in embryonic s tem (ES) cells constructed. Alignment of nucleotide sequences obtained from genomic subclones allowed location of exons 2 through 10 of the alpha(2)-AP gene, but failed to identify the 5' boundary of exon 1. Co mpared to the human gene, exons 2 through 9 in the murine gene have id entical size and intron-exon boundaries obeying the GT/AG rule. The 5' boundary; of exon 10 is identical in both genes while the 3' non-codi ng region is 64 bp longer in the human gene. Introns 2, 3, 6 and 8 hav e similar sizes in the mouse and human genes: intron 1 is 6-fold small er, introns 5, 7 and 9 are 2- to 3-fold smaller, whereas intron 4 is a bout 2-fold larger in the mouse gene. Compared to the human 5' flankin g sequence, an insertion of a simple repeat region with sequence (TGG) n has occurred. The open reading frame of the mouse alpha(2)-AP gene e ncodes a 491-amino-acid protein comprising the experimentally determin ed NH2,-terminus of the mature protein Val-Asp-Leu-Pro-Gly-. A targeti ng vector, pPNT.alpha(2)-AP, was constructed by introducing a homologo us sequence of 8.3 kb in total in the parental pPNT vector. In pPNT.al pha(2)-AP, the neomycin resistance expression cassette replaces a 7 kb genomic fragment comprising exon 2 through part of exon 10 (including the stop codon. which represents the entire sequence encoding the mat ure protein, including the fibrin-binding domain, the reactive site pe ptide bond and the plasmin(ogen)-binding region. Electroporation of 12 9R1 embryonic stem (ES) cells with the linearized vector pPNT.alpha(2) -AP yielded three targeted clones with correct homologous recombinatio n at the 5'- and 3'-ends, as confirmed by Southern blot analysis of pu rified genomic DNA with appropriate restriction enzymes and probes. Th ese targeted clones will be used to generate alpha(2)-AP deficient mic e.