selection of stably transfected cells, although conflicting data about the activity of the CMV promoter in rodent undifferentiated embryonic stem cells (ESCs) have also been obtained [15,16,17,18]. No yellow fluorescent cells were observed (Fig. 3e). These results suggest that at least two phenotypically heteroge- neous cells arise in the HIT-T15 cell culture model. We could not select stably transfected INS-1 823/13 cells because they already contain a pCMV8/INS/IRES/Neo insertion [19]. FACS sorting of the stably transfected HIT-T15 cells followed by q-RT-PCR analysis of each population directly confirmed their heterogeneity with respect to INS gene expression (Fig. 4). High levels of INS transcript were detected in the green population (Fig. 4b), while larger amounts of glucagon (GCG) were detected in the red population (Fig. 4c). Pdx-1 transcripts were expressed at higher levels in GFP+ cells relative to mCherry+ cells (Fig. 4d). The trends were similar, regardless of the reference gene (GAPDH vs. Cyclophilin). FACS-sorted stable GFP+ cells showed much weaker Cre recombinase (red, Cy-3) expression (Fig. 2d) (1–2 copies of the plasmid) relative to the transiently transfected cells (several copies) (Fig. 2b & c), confirming INS+ phenotype. In contrast, FACS- sorted mCherry+ cells did not express Cre recombinase (no green, Cy-2) (Fig. 2d, bottom panel), indicating INS2 phenotype. Furthermore, FACS-sorted GFP+ and mCherry+ cells grown A Dual-Color Reporter for Insulin+ Beta Cells separately in culture maintained their fluorescence throughout five subsequent passages, thus appearing to be stable. These data establish that the color shift from red to green using our reporter system faithfully indicates the status of INS gene expression. They also suggest that the original HIT-T15 population may have been heterogeneous with respect to INS+ phenotype (i.e., a population of GCG+ cells was also present), or perhaps cells change from INS+ to GCG+ in culture by overexpression of aristaless-related homeobox (Arx) (i.e., converting beta-cells into alpha-cells [21,22]). Figure 4e supported the latter case, but we are further characterizing both cell populations by RNA sequencing analysis. Electrophysiological characteristics of FACS-sorted GFP+ and mCherry+ cells We further characterized the FACS-sorted INS-expressing green and non-INS-expressing red cells electrophysiologically. Using a standard whole-cell patch- clamp configuration, we found that the green cells exhibited more than a two-fold greater inward current than the red cells (Fig. 5 a–c; P,0.0001). The kinetics of the inward current were characteristic of Ca2+ channels, an idea further supported by the observation that addition of Cd2+ to the bath effectively blocked the current (Fig. 5d). We also found that a greater proportion of stimulated green cells underwent exocytosis Figure 3. ‘‘Indirect’’ dual-color design of the reporter construct. (a) Transfection of cells with CMV-L-mCherry-L-EGFP. Without Cre recombinase in the construct, all cells exhibited red fluorescence. (b) Schematic and anticipated behavior of pA-Cre-phIns-CMV-L-mCherry-L-EGFP. INS+ beta-cells transfected by this plasmid should activate Cre recombinase expression and excise the mCherry reporter by Cre-LoxP recombination, thus converting red fluorescence to green. In contrast, transfected INS2 non-beta-cells should only exhibit red fluorescence. (c) After transfection of cells with pA-Cre-phIns-CMV-L-mCherry-L-EGFP, HIT-T15 and INS-1 expressed EGFP, indicating that mCherry was excised by Cre recombinase expressed under the human insulin promoter. This demonstrates the reporter’s specificity to produce EGFP in only INS+ cells. (d) Highly magnified (636) projection images of two yellow cells marked as the white square with a white arrow in Fig. 2c from Z-stacks (106), illustrating both red and green fluorescence. In the middle panel, the 3D picture shows co-localization of both colors (B, bottom; T, top). (e) Stable HIT-T15 cell population containing the ‘‘indirect’’ dual-color construct (Neor). ,70% of the stably transfected cells were green, indicating mCherry excision. Another ,10% were red, indicating the absence of mCherry excision and suggesting that the insulin promoter was never activated. The remaining ,20% of cells exhibited no fluorescence, suggesting transcriptional inactivation of the CMV promoter. The right panel depicts the cells after more than 6 days in culture, compared to cells at 1 day after splitting shown in the left panel. No yellow fluorescent cells were observed. These results suggest that at least two phenotypically heterogeneous cells arise in the HIT-T15 cell culture model. doi:10.1371/journal.pone.0035521.g003 PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35521