Characterization and Targeting of the Murine α2-Antiplasmin Gene

Author:

Okada K12,Lijnen H R1,Dewerchin M3,Belayew A1,Matsuo O2,Collen D13,Bernaerts R1

Affiliation:

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

3. The Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium

Abstract

Summaryα2-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.

Publisher

Georg Thieme Verlag KG

Subject

Hematology

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