1. Generation by Homologous Recombination and Characterization

Homozygous plasminogen activator inhibitor-i (PAI-1 )-defi- cient (PAI-1 /-I) mice were generated by homologous recombi- nation in D3 embryonic stem cells. Deletion of the genomic sequences encompassing the transcription initiation site and the entire coding regions ofmurine PAI-i was demonstrated by Southern blot analysis. A 3.0-kb PAI-i-specific mRNA was identified by Northern blot analysis in liver from PAI-1 wild type (PAI-i +1+) but not from PAI-1i-- mice. Plasma PAI-i levels, measured 2-4 h after endotoxin (2.0 mg/kg) injection were 63±2 ng/ml, 30±10 ng/ml, and undetectable (< 2 ng/ ml) in PAl-i++, heterozygous (PA-i +/-) and PAMl-/- mice, respectively (mean±SEM, n = 4-11). PAI-1-specific immunoreactivity was demonstrable in kidneys of PAI-1 +/+ but not of PAl-i-- mice. SDS-gel electrophoresis of plasma incubated with '25I-labeled recombinant human tissue-type plasminogen activator revealed an 115,000-M, component with plasma from endotoxin-stimulated (0.5 mg/kg)PA-i +/+ but not from PAM- -/- mice, which could be precipitated with a polyclonal anti-PAI-1 antiserum. PAM-i- mice were viable, produced similar sizes of litters as PA- +/+ mice, and showed noapparent macroscopic ormicroscopic histological abnormali-