Fig. 4. Clonal analysis of the vascular endothelial lineage and culture of purified endothelial cells. (A) SNV-based, non-replicative retroviral vectors. 5' and 3' long terminal repeats (LTR) are shown as open boxes. An arrow indicates the direction of transcription from the promoter in the left-hand LTR. Bold lines between LTRs denote SNV sequences. Encapsidation sequences required for the packaging of virions are indicated as . Provirus sizes and putative translations of the reporter genes are indicated below the viral structure. (B) Six-day-old embryo inoculated at E4 with a mix of lacZ- and PLAP-carrying vectors. Double histochemistry to reveal the reporter genes and double immunofluorescence with anti-MEP21 (orange) and anti-SMA (blue) mAbs. Cross-section through the aorta. lacZ (black arrowhead) or PLAP (white arrowhead) infections are restricted to the endothelium. (Inset) Two infected cells expressing PLAP framed in B. Scale bars: 50 µm and 20 µm in inset. (C) FACS analysis after AcLDL-DiI and CD45 immunostaining. The frame points to the AcLDL-DiI⁺/CD45^- cell population, i.e. the endothelial cells, selected for culture. (D) Cultures of purified endothelial cells without growth factors (left column), supplemented with VEGF (middle column) or TGFß (right column) at two different time points. Upper line, second day of culture; lower line, fifth day of culture. Triple staining with MEP21 (red)/SMA (green)/DAPI (blue). Two days of culture: purified endothelial cells uniformly express MEP21, although cell sizes in the different culture conditions may vary. Five days of culture: MEP21 expression has decreased and SMA becomes expressed. Disappearance of MEP21 is total in the control medium without growth factors. The VEGF supplementation produces numerous cells co-expressing MEP21 and SMA. With TGFß supplementation, most of the cells have lost MEP21 expression. Some cells, however, retain a MEP21 signal and thus express both markers. Scale bar: 25 µm.