Generation of Plg-A622T mice.

<p>(<b><i>A</i></b>) Structure of the targeted locus in the mouse <i>Plg</i> gene. Exons are represented by <i>filled boxes</i>. A <i>lox</i>P-flanked (<i>filled triangles</i>) neomycin-resistance cassette (<i>NEO</i>) and a diphtheria toxin A fragment expression cassette (<i>DT-A</i>) are indicated by <i>open boxes</i> with <i>arrows</i> that represent the transcriptional orientation. The A622T mutant allele was produced by homologous recombination and <i>NEO</i> deletion mediated by Cre recombinase. The c.1864G>A (p.A622T) mutation and three translationally silent mutations (c.1857T>G, c.1858C>T, c.1860G>A) creating a new <i>Hpa</i>I site (<i>GTTAAC</i>) were introduced into exon 15. Homologous fragments are indicated by <i>dotted lines</i>, while the <i>Mfe</i>I-<i>Hpa</i>I fragments detected by Southern blot analysis of the wild-type (WT) and Plg-A622T alleles are indicated by <i>double-headed arrows</i>. (<b><i>B</i></b>) Southern blot analysis. Genomic DNA from targeted ES cells was digested with <i>Mfe</i>I/<i>Hpa</i>I and detected with the specific probe (WT allele: 9.5 kb; Plg-A622T allele: 6.6 kb). (<b><i>C</i></b>) Quantitative RT-PCR analysis. Total RNA was extracted from mouse liver and subjected to real-time RT-PCR with dual-labeled probes for mouse <i>Plg</i> and <i>Rn18s</i>. Expression levels of <i>Plg</i> mRNA were normalized to <i>Rn18s</i> mRNA. Data are the means ± SDs of WT (n = 8) and <i>Plg</i>^T/T (n = 7) mice. The levels measured in WT mice were defined as 100%.