different levels of expression [13] or unequal catabolism of the two fluorescent proteins [14]. Alternatively, transcriptional inactivation of promoters could have occurred during selection of stably transfected cells [15,16,17,18]. Nevertheless, the presence of the green fluorescent cells complicated clear identification of cells with INS+ versus INS2 phenotypes, so we decided to try a new ‘‘indirect’’ approach. ‘‘Indirect’’ dual-color reporter system Rather than use the insulin promoter to directly drive EGFP expression, we tested an alternate strategy in which the insulin promoter drives expression of Cre recombinase, leading to excision of an mCherry coding region located between CMV promoter and EGFP coding region. The mCherry coding region is followed by a stop codon; thus, the new design leads to mutually exclusive expression of either red or green fluorescent proteins. To confirm that insulin promoter activity led to expression of Cre recombinase, we constructed a plasmid containing the Cre gene under the human insulin promoter (phINS-Cre-pA; Fig. 2a). This plasmid was transiently transfected into HIT-T15 cells, as well as MDA-MB-231 cells (non-insulin-producing negative controls). Roughly 70% of HIT-T15 cells became Cre+ (red), while MB-231 cells did not show Cre expression (Fig. 2b). When transiently transfected with pEGFP-N1, ,70% of cells showed EGFP expression for both cell lines, indicating similar transfection efficiencies (Fig. S1). When HIT-T15 cells were transiently co- transfected with phINS-Cre and pEGFP-N1 (Fig. 2c), ,70% of HIT-T15 cells were Cre+ (red), but not all red cells were green. This suggests that not all cells were transfected with both plasmids and motivated us to use a single construct containing a combination of both cassettes. To make the single construct, we first constructed a plasmid containing only the fluorescent reporters (pCMV-L-mCherry-L- EGFP) (Fig. 3a). This plasmid was transiently transfected into human fibroblasts (a non-insulin-producing negative control), as well as in HIT-T15 cells and the rat insulinoma cell line, INS-1 823/13 [19]. The latter two cell lines are INS+ but should not show any red-to-green color change in the absence of a source of Cre. As expected, all transfected cells showed only red fluorescence (Fig. 3a). We next constructed [pA-Cre-phINS(reverse orientation)]- pCMV-L-mCherry-L-EGFP by inserting a phINS-Cre-pA cas- sette fragment (Fig. 2a) upstream of pCMV-L-mCherry-L-EGFP in reverse orientation (Fig. 3b). Transfected INS+ cells should activate Cre recombinase expression and excise the mCherry reporter by Cre-LoxP recombination, thus converting red fluorescence to green. In contrast, transfected INS– cells should only exhibit red fluorescence. We refer to this reporter as ‘‘indirect,’’ since phINS does not directly drive EGFP expression, but rather changes the color of the reporter being expressed under control of the ubiquitous CMV promoter. Transiently transfected fibroblasts showed only red fluorescence (Fig. 3c), indicating the absence of any Cre-mediated recombina- tion. In contrast, transiently transfected HIT-T15 and INS-1 823/ 13 cells showed either red or green fluorescence, indicating Cre- mediated excision of the mCherry cassette in a subset of the transfected cells. We also observed a small number of HIT-T15 and INS-1 823/13 cells with yellow fluorescence (Fig. 3c), indicating expression of both red and green fluorescent proteins (Fig. 3c). We imaged the cells with confocal microscopy to confirm that the yellow cells showed both green and red fluorescence, rather than being two separate cells stacked atop one another (Fig. 3d, Fig. S2). The yellow fluorescence may be a technical artifact due to unequal copy numbers of our test plasmid in A Dual-Color Reporter for Insulin+ Beta Cells Figure 2. Expression of Cre recombinase from phINS-Cre plasmid. (a) Schematic picture of phINS-Cre construct. (b) Immuno- fluorescence analysis of HIT-T15 and MB-231 cells transiently transfect- ed with phINS-Cre. The red color indicates Cre recombinase expression. Scale bar: 10 mm. (c) Immunostaining of HIT-T15 cells transiently co- transfected with phINS-Cre and pEGFP-N1. The red color indicates Cre recombinase expression (Cy-3), and the green is a marker for co- transfection. (d) Immunostaining of stable HIT-T15 FACS-sorted GFP+ and mCherry+ cells with and without the primary anti-Cre antibody. The green colors in the top and middle panels were visualized as EGFP expression, and the red (Cy-3) as Cre recombinase expression. The stable FACS-sorted mCherry+ cells in the bottom panels did not express Cre recombinase (no green, Cy-2). doi:10.1371/journal.pone.0035521.g002 different cells [13] or unequal intracellular catabolism of red versus green fluorescent proteins [14]. We therefore produced stably transfected HIT-T15 cells using G418 selection for a neomycin resistance gene (Neo) incorporated in our plasmid to present 1–2 copies of the plasmid. We expected all stably transfected cells to exhibit green fluorescence, since HIT-T15 is believed to be a clonal model of beta-like-cells and all should have activated the insulin promoter and expressed Cre [20]. However, we found that only ,70% of the stably transfected cells were green, indicating mCherry excision (Fig. 3e, left panel). 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 that transcriptional inacti- vation of the CMV promoter might have occurred during PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35521