Table 1. Summary of beta-cell-like phenotypes in FACS- sorted HIT-T15 cells. Ca2+ current densitya Extent of exocytosisb Glucose-stimulated increase in [Ca2+]i Insulin gene transcription Glucagon gene transcription Green Cells (GFP+) Larger Larger Yes Yes (high level) Yes (low level) Red Cells (mCherry+) Smaller Smaller No Yes (low level) Yes (High level) aCa2+ current density is measured in pA/pF (current divided by capacitance), a measure of the flow of calcium in the membrane area. GFP+ cells showed 12.661 pA/pF, while mCherry+ cells showed 560.61 pA/pF (p,0.0001). bThe percentage of cells exhibiting capacitance increase was 74% for GFP+ cells and 28% for mCherry+ cells. doi:10.1371/journal.pone.0035521.t001 Table 1 presents a summary of our phenotypic analysis of FACS-sorted stably transfected HIT-T15 cells. The results support our conclusion that our new ‘‘indirect’’ dual color reporter is an effective tool to distinguish a range of beta-cell-like phenotypes. Discussion In the present study, we describe the design and use of a new ‘‘indirect’’ fluorescent reporter system that drives mutually exclusive expression of either EGFP for INS+ cells or mCherry for INS2 cells. This new approach relies on a human insulin promoter-driven Cre-mediated shift in reporter color from red to green in single transgene construct. This results in several advantages over other approaches that use single- or dual-color reporters to distinguish cellular phenotypes. A single-color reporter system with the insulin promoter driving expression of a fluorescent protein [27,28,29] serves to identify a subset of cells fulfilling two conditions: (1) successful transduction/ transfection and (2) activation of the insulin promoter. However, this system does not allow for straightforward calculation of the percentage of transfected/transduced cells. Among those cells that are successfully transfected/transduced, it is not clear what fractions have activated the insulin promoter. The ‘‘direct’’ dual-color reporter systems that have been described use the ubiquitous CMV or a cell-/tissue-specific promoter to drive one color and another cell-/tissue-specific reporter for the second color [30,31,32,33,34,35]. Thus, the cells in which the cell-/tissue-specific promoter is activated should express two colors, while all other cells should express only one color. In principle, this system has the advantage compared to the single-color system of identifying all cells that have been successfully transfected or transduced, so the efficiency of transfection/transduction can be easily calculated. However, a problem with this system became evident when we employed this approach (Fig. 1d & e). For cells in which the tissue-specific promoter had been activated, the relative levels of fluorescence for the two reporter colors was 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 degradation rate of the proteins. In a small subset of cells, the only fluorescence seen was that expected to be due to the activation of the cell-/tissue-specific promoter. It is also possible that in these cells, the CMV promoter was inactivated [15,16,17,18]. A Dual-Color Reporter for Insulin+ Beta Cells The ‘‘indirect’’ dual-color reporter system that we have introduced reports all cells that have been transduced/transfected (so efficiency of transduction/transfection is easily calculated), and it also leads to mutually exclusive marking of cells with one or the other fluorescent protein. Regardless of which fluorescent protein gets expressed, expression is under control of the same strong and ubiquitous 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 (Fig. 3e and Fig. 4b). Another advantage of our indirect dual-color reporter relates to possible off-target effects of the introduced plasmid or viral vector. Introduction of plasmid or viral vectors into cells may alter cellular phenotype [36,37], due to heterologous expression of non-native viral and inserted proteins. Controlling for such changes using a single-color reporter is not possible, since different cultures with control vectors are used to assess phenotypes [29]. In contrast, our approach results in separate colors arising from the same transfected cells using a single transgene construct. The color resulting from the Cre-mediated recombination event (i.e., removal of the mCherry cassette) thus allows GFP+ cells to serve as an appropriate control for non-insulin-positive cell phenotypes. A very similar dual-color system to ours was reported [38], but the investigators used two separate lentiviral constructs that could not eliminate the complexity of cell-to-cell transduction variability ([32], Fig. 2c). Nevertheless, our reporter may need to discern gradients of insulin promoter activity (red-to-green cells over time) in transiently transfected cells using real-time imaging. Using our new reporter, we found that presumably clonal HIT- T15 cells [7,20] were heterogeneous with respect to INS gene expression (Fig. 3e). Approximately 70% of transfected cells were INS+ beta-like-cells, while roughly 30% were INS2, non-beta-like- cells (possibly of mixed cell phenotypes). This result might be due to unanticipated differentiation of the cells into different phenotypic classes during culture or could possibly indicate a non-clonal nature of the reference HIT-T15 cell line we obtained from ATCC. It was recently reported that pancreatic beta-cell identity is maintained by DNA methylation-mediated repression of Arx [21,22]. Deletion of DNA methyl-transferase gene in INS+ beta-cells converts them into GCG+ alpha-cells by derepression of Arx transcription repressor in beta-cells [39,40]. The red population of cells showed a low level of INS and Pdx1 transcript expression and a high level of GCG and Arx transcript expression (Fig. 4), suggesting conversion of beta-cells into alpha-cells, likely by over-expression of Arx through lack of DNA methylation- mediated repression. Regardless, our results argue that care must be taken in interpretation of previous publications on gene expression and functional profiling of HIT-T15 cells [41,42,43] with respect to identification of genes and properties as being exclusive correlates of authentic beta-like phenotypes. Some of the previously reported findings may instead be confounded by the significant population of non-beta-cells. Our fluorescent reporter enables rapid identification and FACS purification of a small percentage of unambiguously identified INS+ beta-like-cells in a mixed population with a significant proportion of non-beta-like-cells (Fig. 4b). We found that GFP+ cells expressed insulin transcript, while mCherry+ cells expressed glucagon (Fig. 4b & c). Furthermore, in comparison to mCherry+ cells, GFP+ cells produced significantly larger inward voltage-gated calcium currents, glucose-stimulated elevation of [Ca2+] , and i larger voltage-stimulated secretion (Fig. 5 and Fig. 6). These characteristics are all indicative of authentic beta-cells [23,44]. In contrast, mCherry+ cells exhibited greater variability in the amplitude of calcium currents and exocytotic responses, perhaps indicating a mixture of different cellular phenotypes in this PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35521