with no insulin promoter activity, the mCherry coding region is not excised, so the cells exhibit red fluorescence. This new ‘‘indirect’’ reporter strategy uses mutually exclusive expression of green or red fluorescence to eliminate ambiguity observed when a human insulin promoter directly drives expression of EGFP in combination with a CMV-regulated mCherry. Distinguishing INS+ from INS- cells with the ‘‘direct’’ strategy depends on identifying cells that are doubly fluorescent and often leads to ambiguous results—the relative levels of fluorescence for the two reporter colors can be highly variable (due to variability in the relative strength of the two promoters driving fluorescent protein expression, differences in the relative fluorescence intensities, and/ or relative rate of degradation of the proteins). Our ‘‘indirect’’ dual-color system, in contrast, reports all cells that have been transduced or transfected, so efficiency of transduction/transfec- tion is easily calculated. Regardless of which fluorescent protein is expressed, expression is under control of the same CMV promoter. We thus observe only a single color for each phenotype that reports successful Ins gene expression by a discrete change in color from red to green. In addition, our approach results in separate colors arising from the same transfected cells using a single transgene construct, which is not possible with a single-color reporter. We prepared HIT-T15 cells stably transfected with our new reporter construct. Contrary to expectations, we observed that these presumably clonal cells are in fact heterogeneous with respect to a variety of beta-cell-like biochemical and electrophys- iological phenotypes. After FACS isolation of green fluorescent cells, we obtained a stable, homogeneous population of insulin- producing cells with beta-like phenotypes. Since our reporter system uses the human insulin promoter, we anticipate its utility to identify and FACS purify INS+ and glucose-responsive beta-like- cells from primary human islet cell isolates or in vitro differentiated human pluripotent stem cells. A Dual-Color Reporter for Insulin+ Beta Cells Results Specificity of the proximal 378-nt region in the human insulin promoter The complete human insulin promoter/enhancer is estimated to be ,4 kb in length [8,9]. The proximal 378-nt region (2363 to +15) is highly conserved between humans and rodents [10,11]. It contains important regulatory motifs (Fig. 1a) previously used to make reporters for identifying INS+ cells [6,12]. We first confirmed the utility of this region to report insulin production by transfecting a phINS-EGFP construct (Fig. 1b, [6]) into the HIT-T15 hamster insulinoma clonal cell line (insulin-producing) [7], as well as the NT-2 human embryonic carcinoma cell line (non-insulin-producing). As expected, only HIT-T15 cells exhib- ited green fluorescence (Fig. 1c). In contrast, using the ubiquitous CMV promoter instead of the insulin promoter to drive EGFP expression resulted in fluorescence of both cell types (Fig. 1c). We therefore concluded that this 378-nt promoter region is sufficient for marking insulin-producing cells, and we used this region to construct additional reporters. ‘‘Direct ’’ dual-color reporter system We modified phINS-EGFP by introducing a dsRed reporter cassette under control of the CMV promoter, reasoning that this could be used to control for transfection efficiency and to identify non-insulin producing cells. We expected INS2 cells to exhibit only red fluorescence, and INS+ cells to exhibit both red and green (yellow) fluorescence. Transient (data not shown) and stable transfection of phINS-EGFP-CMV-dsRed (Fig. 1d) into HIT-T15 cells resulted in cells that displayed red or yellow fluorescence (Fig. 1e). However, a small population of cells unexpectedly appeared to express primarily green fluorescence (Fig. 1e). We reasoned that this unexpected fluorescence was caused by unequal accumulation of fluorescent reporter protein in certain cells with Figure 1. Specificity of the human insulin promoter (378-nt region) to identify insulin-producing cells and a direct dual-color reporter system. (a) Schematic picture of the proximal 378-nt region (2363 to +15) of the human insulin promoter containing several regulatory motifs for expression of the insulin gene. (b) The phINS-EGFP construct [6] used to express EGFP in insulin-producing cells. (c) phINS-EGFP was transfected into HIT-T15 or NT-2 cells. After 2 days, some HIT-T15 cells expressed EGFP, but NT-2 cells did not. When pCMV-EGFP was transfected, both cell lines expressed EGFP, suggesting the specificity of the 378-nt region to identify insulin-producing cells. (d) Schematic picture of a direct dual-color reporter that contains a human insulin promoter driving expression of EGFP and a CMV promoter driving expression of dsRed (phINS- EGFP-CMV-dsRed). (e) Stable transfection of phINS-EGFP-CMV-dsRed into HIT-T15. Many cells displayed red or yellow fluorescence, but a small population of cells unexpectedly appeared to express primarily green fluorescence (white arrows). doi:10.1371/journal.pone.0035521.g001 PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35521