Evaluation of TH-eGFP promoter constructs in mouse embryonic stem (mES) cells during in vitro differentiation. (A, B): Overlap in eGFP and TH expression in a (A) 2.5-kilobase (kb) TH-GFP clone and in a (B) 9-kb TH-eGFP clone after 14 days of differentiation on PA6. Cultures of 9-kb TH-eGFP clones contained many more eGFP+/TH+ cells compared with the 2.5-kb TH-GFP cultures (yellow co-expression). (C): FACS analysis for eGFP+ events within an FSC/SSC gate of naïve D3 mES cells autofluorescence), one 2.5-kb TH-eGFP clone (number 91) and three 9-kb TH-eGFP clones (numbers 15, 41, and 93) after 9 days of differentiation on PA6. All 9-kb TH clones showed an expected higher proportion of eGFP+ events compared with the naïve D3 mES cell line (*, p < .01 to p < .001; analysis of variance [ANOVA]) and to the 2.5-kb clone (#, p < .01 to p < .001; ANOVA). The 2.5-kb TH-eGFP clone was not significantly different from the D3 cells (*, p < .05 to p < .001; ANOVA). Scale bars = 50 μm (A, B). Abbreviations: eGFP, enhanced green fluorescent protein; FSC, forward scatter; GFP, green fluorescent protein; k, kilobase; SSC, side scatter; TH, tyrosine hydroxylase.