Engineering ESCs expressing Venus-GFP under the regulatory region of Mesp1. (A) Schematic representation of the Mesp1 reporter transgene. Venus-GFP is cloned under the regulatory sequences of Mesp1 that allowed transgene expression in the cardiogenic mesoderm. (B, right) Detection of GFP in Mesp1-GFP ESCs at D3 of differentiation. (left) Unmodified ESCs at the same day of differentiation are used as a control. Bars, 50 µm. (C and D) Kinetics of Mesp1 mRNA expression measured by RT–quantitative PCR (C), and Mesp1-GFP expression as detected by FACS (D). Results are normalized for Mesp1 expression in undifferentiated ESCs (C) or represent the percentage of Mesp1-GFP–positive cells (D). (E) Relative expression of Mesp1 and GFP transcripts in Mesp1-GFP–expressing cells (GFP positive [pos]) and in Mesp1-GFP–nonexpressing cells (GFP negative [neg]) isolated by FACS at D3. Results are normalized for the expression of the transcripts in all sorted cells (gray bars). Error bars indicate means ± SEM; n = 3. FRT, flippase recognition target. pA, polyadenylation. PGK, phosphoglycerate kinase.