Gene targeting and visualization of transcription at the Tmtsp locus. (A) Schematic illustration of gene targeting construct. DNA sequence corresponding to 2 translational start codons was replaced by Venus-Neo cassette. Components of the cassette were as follows: splicing donor, D; splicing acceptor, A; enhanced mutant of yellow fluorescent protein, Venus; simian virus 40 (SV40)–derived polyadenylation signal, pA; and phosphoglycerate kinase promoter-neomycin phosphotransferase, PGK-Neo. Numbered black boxes indicate exons. Recognition site of EcoNI (E) and HindIII (H) are shown by triangles. (B) Southern blot analysis of mutant mouse. Genomic DNA digested with EcoNI and HindIII were hybridized with 5′ and 3′ probes, respectively (shown in A). (C) Image of EBs (day 4) from knock-in ES cells. (D) Correlation of Venus signal and Tmtsp protein. EBs (day 7) from knock-in ES cells were stained with anti-Tmtsp antibody and analyzed by flow cytometry. Tmtsp protein was expressed only on Venus⁺ (Venus^low to Venus^high) cells. (E) Immunostaining of endothelial cells on OP-9 stromal cells. After 4 days of EB formation, cells were replated on OP-9 cells and cultured for 3 days in the presence of VEGF (stained with anti-Tmtsp, VE-cadherin, and CD31/PECAM-1) or in the presence of VEGF-D (for lymphatic endothelium formation, stained with anti–LYVE-1 antibody). (F) Whole-mount immunostaining of E10.5 knock-in embryo. (i) Overview of E10.5 embryo. (ii) Venus signal (green) was highly detected in vascular network of the embryo body. (iii) Overlaid image of Venus signal and immunostaining with CD31/PECAM-1 showed expression of Venus in the capillary network beneath the body surface. Scale bar: 50 μm.