A Novel Dual-Color Reporter for Identifying Insulin- Producing Beta- Cells and Classifying Heterogeneity of Insulinoma Cell Lines Nan Sook Lee1*, Joyce G. Rohan1¤a, Madison Zitting2, Sonia Kamath1, Andrew Weitz2, Arnold Sipos3, Paul M. Salvaterra4, Kouichi Hasegawa5¤b, Martin Pera5¤c, Robert H. Chow1 1 Department of Physiology & Biophysics and Zilkha Neurogenetics Institute, University of Southern California, Los Angeles, California, United States of America, 2 Department of Biomedical Engineering, University of Southern California, Los Angeles, California, United States of America, 3 School of Medicine, University of Southern California, Los Angeles, California, United States of America, 4 Division of Neuroscience, Beckman research Institute of the City of Hope, Duarte, California, United States of America, 5 Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of Southern California, Los Angeles, California, United States of America Abstract Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use of a novel ‘‘indirect’’ dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin- producing (INS+) beta-cells or mCherry in non-beta-cells. Our system uses the human insulin promoter to initiate a Cre- mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS+ phenotype. Our strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of INS+ and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem cells. Citation: Lee NS, Rohan JG, Zitting M, Kamath S, Weitz A, et al. (2012) A Novel Dual-Color Reporter for Identifying Insulin-Producing Beta- Cells and Classifying Heterogeneity of Insulinoma Cell Lines. PLoS ONE 7(4): e35521. doi:10.1371/journal.pone.0035521 Editor: Francis C. Lynn, University of British Columbia, Canada Received December 6, 2011; Accepted March 17, 2012; Published April 18, 2012 Copyright: 2012 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Wright Foundation (USC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nanlee@usc.edu ¤a Current address: Department of Neuroscience, Cell Biology and Physiology, Wright State University, Dayton, Ohio, United States of America ¤b Current address: Institute for Integrated Cell-Material Science, Kyoto University, Kyoto, Japan ¤c Current address: The Stem Cell Program, University of Melbourne, Melbourne, Australia Introduction Diabetes prevalence is increasing dramatically worldwide and severe co-morbidities persist, despite the availability of insulin treatment. Cell replacement strategies are thus being developed to treat this metabolic disease. A key treatment step will be production of sufficient quantities of fully functional pancreatic beta- or beta-like-cells suitable for replacing missing or defective beta-cells. This goal has stimulated renewed interest in under- standing human islet cell biology. However, because of the difficulty and high cost associated with isolating human islets, most studies focus on in vitro characterization of immortalized human or animal cell lines as surrogates for primary beta-cells. Rodent insulinoma cell lines derived from cancers arising after radiation treatment (rat RIN, INS-1, CRI-G1) or viral transfor- mation (hamster HIT, bHC) have been especially useful models of beta-cell biology; at the time of their establishment, they exhibit high levels of insulin production and glucose respon- siveness [1,2,3,4]. However, both of these pancreatic beta-cell attributes are lost with time in culture and increased numbers of cell passages [5]. Unfortunately, the generation and characteriza- tion of human insulinoma or beta-cell-derived cell lines that preserve normal glucose responsiveness has not been reported. The hamster insulinoma cell line, HIT-T15, has been one of the most extensively studied beta-cell-like models. HIT-T15 cells exhibit glucose-stimulated insulin secretion and contain mem- brane-bound secretory granules [6], similar to those seen in normal islet beta-cells. HIT-T15 cells were originally produced by SV40 transformation of pancreatic beta-cells, followed by serial selection of clonal lines expressing the glucose-responsive phenotype [7]. In this study, we describe the development and application of a new dual-color fluorescent reporter system for identifying insulin- producing (INS+) beta- and non-insulin-producing (INS-) cells. Our reporter contains a single transgene with two expression cassettes. The first is a fragment of the human insulin gene promoter (phINS) that drives expression of Cre recombinase protein exclusively in INS+ cells. The second contains the CMV promoter and an mCherry coding region flanked with LoxP (L) sites, followed by an EGFP coding region. In cells with active insulin promoter activity, the Cre protein excises the mCherry coding region, and the cells exhibit green fluorescence. In cells PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35521