Fig 1. Generation of Plg-A622T mice.
(A) Structure of the targeted locus in the mouse Plg gene. Exons are represented by filled boxes. A loxP-flanked (filled triangles) neomycin-resistance cassette (NEO) and a diphtheria toxin A fragment expression cassette (DT-A) are indicated by open boxes with arrows that represent the transcriptional orientation. The A622T mutant allele was produced by homologous recombination and NEO 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 HpaI site (GTTAAC) were introduced into exon 15. Homologous fragments are indicated by dotted lines, while the MfeI-HpaI fragments detected by Southern blot analysis of the wild-type (WT) and Plg-A622T alleles are indicated by double-headed arrows. (B) Southern blot analysis. Genomic DNA from targeted ES cells was digested with MfeI/HpaI and detected with the specific probe (WT allele: 9.5 kb; Plg-A622T allele: 6.6 kb). (C) Quantitative RT-PCR analysis. Total RNA was extracted from mouse liver and subjected to real-time RT-PCR with dual-labeled probes for mouse Plg and Rn18s. Expression levels of Plg mRNA were normalized to Rn18s mRNA. Data are the means ± SDs of WT (n = 8) and PlgT/T (n = 7) mice. The levels measured in WT mice were defined as 100%.