Establishment of transgenic mice expressing u-PA in their platelets. (A) Schematic representation of the 10.2-kb Sac II insert containing the 1.2-kb murine XbaI/KpnI PF4 promoter (open box) plus 5′-UTR (light gray box) followed by the 11-exon murine u-PA gene (black boxes) and ending with the hGH 3′-UTR and polyadenylation sequence (dark gray box). The 2.8-kb Bgl II fragment detected for the transgene in the genomic Southern blot in panel B is indicated. PCR was performed to confirm founder lines using primers complementary to the mouse PF4 5′-UTR and exon 2 of the murine u-PA gene (data not shown). The same primers were used for the RT-PCR in panel C indicated as open arrows. (B) Genomic Southern blot analysis of Bgl II-digested DNA from a wild-type animal and the 3 lines that had offspring is shown. Copy number determined by phosphorimaging is indicated below. (C) RT-PCR of platelet total RNA for the 114-bp transgenic u-PA message is shown, as is the 185-bp PF4 message as a positive control of the platelet nature of the RNA. - indicates RT-PCR; D, pretreatment with RNase-free DNase prior to RT-PCR; R, pretreatment with DNase-free RNase prior to RT-PCR; H, water used instead of first-strand cDNA. Arrows point to the expected cDNA bands. (D) RT-PCR of multiple tissues. Only bone marrow showed the 114-bp transgenic u-PA band, but all samples showed the 249-bp HPRT⁺ control cDNA band. 1 indicates spleen; 2, liver; 3, lung; 4, heart; 5, kidney; 6, adrenal; 7, tongue; 8, brain; 9, bone marrow. Arrows point to the expected cDNA bands. (E) Immunohistochemical detection of u-PA in wild-type and line no. 19 bone marrow. Several morphologically recognizable megakaryocytes are indicated by arrows. Original magnification, × 1000; samples were stained with hematoxylin.