The Eng promoter when combined with the −8, +7, and +9 hematoendothelial enhancers targets FLK1+ mesodermal cells enriched for BL-CFC potential. (A) Schematic representation of the experimental procedure. The Eng −8/P/LacZ, Eng −8/P/LacZ/+7, Eng −8/P/LacZ/+9, Eng −8/P/LacZ/+7/+9, Eng −8/P/LacZ/+7Δ/+9, and Eng −8/P/LacZ/+7Δ/+9Δ reporter constructs were introduced by homologous recombination into the HPRT locus of HM1 ES cells. Recombinant clones were differentiated into day 3 EBs and stained for FLK1 expression and β-galactosidase activity. FLK1+/LacZ− (F+/L−; gray) and FLK1+/LacZ+ (F+/L+; blue) cells were sorted and seeded into BL-CFC assays. Fractions sorted from the Eng −8/P/LacZ/+7/+9 were further analyzed by RNA sequencing. (B) Flow cytometry profiles of Eng −8/P/LacZ/+7/+9 day 3 EBs (left). BL-CFCs from sorted F+/L− (gray) and F+/L+ (blue) fractions. (C) (i) Flow cytometry profile of day 3 EBs derived from ES cells targeted with Eng −8/P/LacZ/+7Δ/+9 (mutated GATA motifs in the +7 enhancer) is shown to the left with corresponding BL-CFCs from sorted F+/L− (gray) and F+/L+ (blue) fractions shown to the right. (ii) Flow cytometry profile of day 3 EBs derived from ES cells targeted with Eng −8/P/LacZ/+7Δ/+9Δ (mutated GATA motifs in the +7 enhancer and mutated ETS motifs in the +9 enhancer) and corresponding BL-CFCs from sorted F+/L− (gray) and F+/L+ (blue) fractions. (D) Flow cytometry profiles of day 3 EBs and BL-CFCs from sorted F+/L− (gray) and F+/L+ (blue) fractions are shown for ES cells targeted with (i) Eng −8/P/LacZ, (ii) Eng −8/P/LacZ/+9, and (iii) Eng −8/P/LacZ/+7. BL-CFC counts are the total number of blast colonies generated from 2 × 10⁴ seeded cells. Statistical analysis was conducted using Student t test, *P < .05, **P < .01.