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.