population. In the future, we will use our reporter to isolate purely homogeneous beta- or beta-like-cells, which will later be used for more accurate genetic, epigenetic, and functional profiling. Our approach should also allow us to compare gene expression and functional phenotypes of beta- or beta-like-cells with other types of pancreatic cells that may be present in the mCherry+ class of cells (a, d, and PP cells, or perhaps even exocrine acinar cells). Human islet transplantation using the Edmonton protocol is an effective treatment for type I diabetes [26]. Its widespread applicability, however, is limited because of a scarcity of donor tissue. Properly and correctly differentiated beta-cells from human pluripotent stem cells could potentially overcome this limitation. Most current human pluripotent stem cell differentiation protocols [45,46] have limited reproducibility, low yield of beta-like-cells, and most importantly, the absence of glucose responsiveness [45]. We expect that our reporter will facilitate easy and quick evaluation of new differentiation protocols designed to produce clinically useful beta-cells—it could be stably introduced into human pluripotent stem cells [47] or transduced into different stage cells using an adenoviral vector [48,49]. In addition, the reporter system may be used as a high-throughput screening for reprogramming non-insulin-producing cells to insulin-producing cells. Further additions and modifications to our approach could be easily incorporated, depending on the particular phenotypic property desired. For example, other promoters could be used to mark different cell- or tissue-specific lineages and to further test their homo/heterogeneity. Materials and Methods Cell culture, transfection, FACS sorting and q-RT-PCR HIT-T15 [6] (ATCC, USA), INS-1 823/13 (a gift from Chris Newgard [19]) and NT-2 cells [50] were cultured as descried previously. Human dermal fibroblasts (ScienCell) were cultured in MEM alpha medium (Invitrogen) with 0.1 mM MEM nonessen- tial amino acid, 10% FBS, and penicillin/streptomycin. MDA- MB-231 cells were cultured in DMEM medium with 10% FBS and penicillin/streptomycin. All cells were incubated at 37uC with 5% CO2 in a humidified incubator. For all transfection experiments, unless otherwise specified, we used Lipofectamine 2000 (Invitrogen) using the manufacturer’s instructions. For making stable HIT-T15 cells, cells were transiently transfected with our reporter (pA-Cre-pINS-pCMV-loxP-mCherry-pA-loxP- EGFP-pA). Two days after transfection, cells were split at a 1:10 ratio with fresh growth medium into 10 cm tissue culture dishes and selected with 800 mg/ml G418 until colonies were visible. The whole populations of stable transfectants or individual colonies were screened for expression of fluorescent proteins. Expression of fluorescent proteins was examined with an Olympus IX70 fluorescence microscope (Olympus, Japan) or a Leica TCS SP5 confocal microscope (Leica Microsystems, Germany). Images were analyzed and edited in MetaMorph software. For cell sorting, cells were trypsinized after imaging, suspended in medium, and subjected to FACS sorting for GFP+ and mCherry+ cells. FACS was performed by the USC FACS Core Facility, using a BD FACSAria cell sorter. After cell sorting, total RNAs were isolated from GFP+ or mCherry+ cells using Trizol (Invitrogen). After digestion of total RNAs with Turbo DNase (Ambion), cDNAs were made from the total RNAs with an iScript cDNA synthesis kit (Bio-Rad), according to the manufacturer’s instructions. The cDNA was diluted three-fold with water prior to quantitative PCR (q-PCR) analysis. Gene-specific q-PCR primer/ probe sets (customized TaqMan Gene Expression Assays, Applied A Dual-Color Reporter for Insulin+ Beta Cells Biosystems) for hamster INS, GCG, GAPDH, Cyclophillin, and human Pdx-1 and equivalent amounts of cDNA generated as a template were used for q-PCR. Reactions were performed in for each sample using TaqMan Universal PCR Master Mix with a CFX-96 system (Bio-Rad). The q-PCR was performed using the manufacturer’s instructions. For each sample, expression of marker genes was normalized to GAPDH or Cyclophillin. Data are expressed as a relative expression level. For detection of Arx that is not well studied in the hamster system, we performed q- PCR using mouse primers (F:59-TTCCAGAAGACGCAC- TACCC; R: 59-TCTGTCAGGTCCAGCCTCAT [21]) as de- scried previously [50]. Immonofluorescent staining and confocal microscopy Cells grown on sterilized, 0.4% gelatin-coated patch-clamp dishes were washed with PBS and fixed with 4% paraformalde- hyde for 20 minutes at room temperature. For detection of Cre nuclear proteins, dishes were permeabilized for 10 minutes with 0.25% Nonidet P-40 (NP40). Cells were blocked overnight at 4uC with 5% fetal goat serum, 1% bovine serum albumin, and 0.1% Triton X-100, and incubated for 1 hour with the primary mouse monoclonal anti-Cre antibody (Sigma-Aldrich, a gift from Le Ma) diluted in blocking solution (1:500). Cells were then washed and incubated for 1 hour with the secondary anti-mouse Cy-2 or Cy-3 conjugated antibody (1:250; Jackson ImmunoResearch, a gift from Le Ma). Images were taken using a Leica TCS SP5 confocal microscope. EGFP and mCherry were visualized by endogenous fluorescence. Fluorescent signal of EGFP and mCherry in yellow cells were visualized by a Leica TCS SP2 AOBS confocal microscope system (Leica-Microsystems, Heidelberg, Germany) using 106 and 636 magnifications. A Leica DM IRE2 inverted microscope was powered by Argon and HeNe lasers for the detection of EGFP (excitation at 488 nm, emission at 495–550 nm) and mCherry (excitation at 594 nm, emission at 600–750 nm) fluorescence. Images were collected in xyz series and analyzed by Leica LCS imaging software (LCS 3D, Process, and Quantify packages). DNA constructs and expression All constructs were made using standard molecular cloning methods. To construct phINS-EGFP-pA-pCMV-DsRed2-pA, plasmid phINS-EGFP [6] was digested using SalI and blunted at both sites with Klenow DNA polymerase (NEB), obtaining phINS- EGFP-pA fragment. The fragment was then subcloned upstream of CMV promoter in pDsRed2-N1 (Clontech) digested using AseI and blunted at both sites with Klenow. For constructing pCMV-L-mCherry-pA-L-EGFP-pA, a frag- ment of L-mCherry-pA-L containing HindIII and XhoI restriction enzyme sites at each 59 and 39 ends, respectively, was obtained by polymerase chain reaction (PCR) using a template (lox2272- mCherry-loxP in pBlue-script SK, a gift from Li Zhang [51]), and forward (59-catgggaagcttataacttcgtatagcatacattatacgaagttatGC- CACCATGGTGAGCAAGGG- CGAGGA) and reverse (59- ggaattcctcgagataacttcgtataatgtatgctatacgaagttatAGATC- TGTCGAGGCCGCGAATTAAAAAACC) primers. Underlined sequences are the LoxP site and its reverse complement, and the bold capitalized ATG is the start codon of mCherry. The bold fonts are the restriction enzyme sites for digestion. The PCR product was cut by HindIII plus XhoI and blunted at the XhoI site with Klenow. The HindIII-XhoI (blunt) fragment was inserted into pEGFP-N1 (Clontech) digested with HindIII plus SalI and blunted at SalI site with Klenow. To obtain plasmid phINS-Cre-pA, a Cre-pA fragment was generated by PCR using pCMV-Cre-pA (a gift from Li Zhang) as PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35521