Characterization and Targeting of the Murine α₂-Antiplasmin Gene

Summary

α₂-Antiplasmin (α₂-AP) is the main physiological plasmin inhibitor in mammalian plasma. As a first step toward the generation of α₂-AP deficient mice, the murine α₂-AP ₁ gene was characterized and a targeting vector for homologous recombination in embryonic stem (ES) cells constructed. Alignment of nucleotide sequences obtained from genomic subclones allowed location of exons 2 through 10 of the α₂-AP ₁gene, but failed to identify the 5’ boundary of exon 1. Compared to the human gene, exons 2 through 9 in the murine gene have identical 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-coding region is 64 bp longer in the human gene. Introns 2,3,6 and 8 have similar sizes in the mouse and human genes; intron 1 is 6-fold smaller, introns 5, 7 and 9 are 2- to 3-fold smaller, whereas intron 4 is about 2-fold larger in the mouse gene. Compared to the human 5’ flanking sequence, an insertion of a simple repeat region with sequence (TGG)n has occurred. The open reading frame of the mouse α₂-AP gene encodes a 491-amino acid protein comprising the experimentally determined NH₂-terminus of the mature protein Val-Asp-Leu-Pro-Gly-.

A targeting vector, ppPNT.α₂-AP, was constructed by introducing a homologous sequence of 8.3 kb in total in the parental pPNT vector. In pPNT.α₂-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 mature protein, including the fibrin-binding domain, the reactive site peptide bond and the plasmin(ogen)-binding region. Electroporation of 129R1 embryonic stem (ES) cells with the linearized vector pPNT.α₂-AP yielded three targeted clones with correct homologous recombination at the 5’- and 3’-ends, as confirmed by Southern blot analysis of purified genomic DNA with appropriate restriction enzymes and probes. These targeted clones will be used to generate α₂-AP deficient mice.

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