Fig. 4. Selection of endothelial cells from a population of differentiating tie-1-EGFP/tie-1-puro^r ES cells by puromycin. (A) Analysis of tie-1-puro^r transgene integration into genomic DNA. PCR products were amplified using oligonucleotides for the tie-1 promoter and either puro^r gene or the EGFP cDNA. PCR products are shown for two resistant clones, B9TP and C4TP, and the parental clone A1TG. Amplification of a 500 bp fragment of the tie-1-puro^r transgene in B9TP (lane 2), C4TP (lane 3) and with the tie-1-puro^r control plasmid (lane 4). Amplification of a 310 bp fragment of the tie-1-EGFP transgene in the parental clone A1TG (lane 5), B9TP (lane 6), C4TP (lane 7) and with the tie-1-EGFP control plasmid (lane 8). Lane 1, molecular weight markers. (B) Puromycin selection of ES-derived endothelial cells from 2D plane cultures of B9TP cells. B9TP cells were allowed to differentiate for 7 days in the presence of 10 ng/ml rVEGF and subjected (right panel) or not (left panel) to a 4-day puromycin selection (1 µg/ml). While in the absence of puromycin, CD31⁺EGFP⁺ cells are present in an heterogenous population, after puromycin selection, almost all remaining cells are CD31⁺EGFP⁺. DAPI staining allows visualization of all cells present in the field. Bar, 100 µM.