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Thromb Haemost 1997; 78(03): 1104-1110
DOI: 10.1055/s-0038-1657694
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Characterization and Targeting of the Murine α2-Antiplasmin Gene

K Okada
1   The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
2   The Department of Physiology, Kinki University School of Medicine, Osaka, Japan
,
H R Lijnen
1   The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
,
M Dewerchin
3   The Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium
,
A Belayew
1   The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
,
O Matsuo
2   The Department of Physiology, Kinki University School of Medicine, Osaka, Japan
,
D Collen
1   The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
3   The Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium
,
R Bernaerts
1   The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
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Publikationsverlauf

Received 19. 1996

Accepted after revision 08. April 1997

Publikationsdatum:
12. Juli 2018 (online)

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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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