Generation of the Plau^GFDhu/GFDhu mouse strain. (A) Structure of targeting vector (top), wild-type Plau allele (middle), and targeted Plau allele with the neomycin cassette removed (bottom). Exons are indicated as blue boxes and intron sequences as black lines. Homologous recombination in ES cells introduced c.185T→A, c.201G→A, c.202A→C, c.207G→A, c.209C→T, and c.211A→G mutations into exon 4 and a neomycin selection cassette (orange) flanked by LoxP sites (red triangles) into intron 4. The neomycin cassette was removed by crossing germline chimeras to the Oz_Cre deletor strain (Ozgene Pty). The locations of the primers used for PCR screening of ES cell clones and genotyping of mice are indicated by arrows. NNHW indicates the position of the 4 codon changes in exon 4. Solid green box denotes location of Southern blot probe. Position of EcoRV restriction sites (RV) are indicated. (B) Southern blot of EcoRV digested tail DNA from Plau^+/GFDhu (lane 1), Plau^+/+ (lane 2), and Plau^+/GFDhu-neo (lane 3) offspring of a Plau^GFDhu/+ germline chimera crossed to an Oz_Cre deletor strain mouse. Positions of wildtype, targeted allele with the neomycin cassette, and targeted allele with the neomycin cassette removed are shown. Position of molecular weight markers (kb) are indicated at left. (C) Structure of the mouse uPA GFD. Combined surface (semitransparent), ribbon, and stick representation depicts the receptor-binding β-hairpin of the GFD of mouse uPA using the atomic coordinates 3LAQ¹⁷  and Pymol (DeLano Scientific). The 4 mutated amino acids (Y23N, R28N, R30H, R31W) are shown as sticks. (D) Sequence analysis of part of exon 4 (c.179-217) from Plau^+/+ (top) and Plau^GFDhu/GFDhu (bottom) mice confirms the introduction of the 6 nucleotide substitutions and 4 amino acid substitutions (red letters in nucleotide and amino acid sequence). (E) qPCR analysis of total RNA from Plau^+/+ (left), Plau^GFDhu/GFDhu (middle), and Plau^−/− (right) mouse kidneys shows that Plau mRNA levels are not affected by the mutations in exon 4 or the LoxP site in intron 4. Data are shown as mean ± SEM, 3 mice per genotype. (F) Western blot of uPA in pooled samples (3-4 mice per group) of void urine from Plau^+/+ (left), Plau^−/− (middle), and Plau^GFDhu/GFDhu (right) shows normal expression of high-molecular-weight (HMW, arrow) uPA^{Y23N-R28N-R30H-R31W}, reduced LWM (arrow) uPA^{Y23N-R28N-R30H-R31W}, and a corresponding increase in a higher-molecular-weight form processed in the linker region between the GFD and kringle domains. The position of molecular weight markers (kDa) is indicated at left.