Enhancer Function of Integrated Alb Reporters Figure 3. Transient transfection vs. stable gene integration for determining enhancer activity. A: Plasmid constructs. Plasmid P contains the 0.2 kb promoter region fused to GFP. Plasmid 1+2+P also contains a 17.4 kb contiguous upstream segment (Regions 1 and 2). The dotted line represents the vector backbone. B: Transient transfection assays. Upper panel,scatter plots; lower panel, histogram subtraction. The solid green curve is the calculated fluorescence due to transfection. C: Plot of mean fluorescence intensity for each plasmid corrected for molar plasmid concentration and normalized to the promoter value. Average and standard deviation of 3 separate transfection experiments, each with triplicate transfections. D: Cell images. The promoter region produced weak gene expression that was strongly stimulated by the added enhancer region. The images show Orientation 1 integrants. E: Flow cytometric analysis of stable clones showing orientation-dependent stimulation by enhancers. Control cells, open curve; P, gray; 1+2+P, black. F: Expression in HuH7-9 cells. G: Expression in HuH7-10 cells. F, G; Plots show the average and standard deviation of fluorescence from three clones in each orientation. O1, O2; orientations 1 and 2. doi:10.1371/journal.pone.0023234.g003 PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23234