The study was approved by the Ethics Committee of Chungbuk National University (Cheongju, Korea). The porcine fibroblasts were obtained from miniature pig fetuses (Yucatan pigs; Optifarm Solution Inc., Gyeonggi-do, Korea) on the thirtieth day of pregnancy and the cells were routinely maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 25 mM glucose, supplemented with 10% fetal bovine serum (FBS; WelGENE, Inc., Daejeon, South Korea), 100 U/ml penicillin and 100 μg/ml streptomycin. The mouse β-cell line, MIN6 (American Type Culture Collection, Manassas, VA, USA), was cultured in DMEM containing 25 mM glucose, supplemented with 15% FBS, 55 μM 2-mercaptoethanol (Gibco, Grand island, NY, USA), 100 U/ml penicillin and 100 μg/ml streptomycin. All cells were grown in a humidified 5% CO₂ atmosphere at 37°C. Unless otherwise indicated, all cell culture materials were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA).

Genomic DNA from the cells was isolated with a G-DEX™ IIc Genomic DNA Extraction kit (iNtRON Biotechnology, Inc., Seoul, South Korea). Genomic DNA (0.1 μg) was amplified in a 20-μl PCR reaction containing 1 U i-Start Taq polymerase (iNtRON Biotechnology, Inc.), 2 mM dNTPs (Takara Bio, Inc., Shiga, Japan) and 10 pmol of each specific primer. The details of all primers are described in Table I. The PCR reactions were denatured at 94°C for 30 sec, annealed at 62°C for 30 sec and extended at 72°C for 1–2 min. The PCR products were subjected to cloning processes and/or separated on a 1% agarose gel, stained with ethidium bromide and photographed under UV illumination. The image was scanned using GelDoc EQ (Bio-Rad, Hercules, CA, USA).

All restriction enzymes were obtained from Takara Bio, Inc. The human insulin promoter region (from nucleotides (nt) −1,431 to +1 nt; +1 = the transcriptional start site) demonstrated the highest transcriptional activity in a previous study (14) and were prepared by long-range PCR using human genomic DNA (Clontech Laboratories, Mountain View, CA, USA) as the template, and specific primers containing restriction enzyme sites (SpeI at the 5′ end or EcoRI at the 3′ end). Amplified fragments were digested with SpeI and EcoRI and replaced with the CMV promoter of the transcriptional activator construct, pCMV-Tet3G, purchased from Clontech Laboratories. The ICER Iγ cDNA was prepared by PCR using genomic DNA from pig pancreas as the template and was inserted into pTRE3G-ZsGreen1 which contains green fluorescence protein, ZsGreen1 (Clontech Laboratories) through MluI at the 5′ end or BamHI at the 3′ end. Using the PCR method, the ICER Iγ-expressing tTA-response region obtained from recombinant pTRE3G-ZsGreen1-pig ICER Iγ construct was digested with Bst1107I or SpeI and combined with the recombinant pHINSP-Tet3G vector controlled by the human insulin promoter.

The fibroblasts were transfected with the linearized targeting vector using Lipofectamine^® 2000 (Invitrogen, Carlsbad, CA, USA). Following 24 h of transfection, the medium was replaced with DMEM supplemented with 10% FBS and 250 μg/ml G-418 (Roche Diagnostics, Indianapolis, IN, USA) for four weeks. The antibiotic-resistant cells were further selected, subjected to PCR-based genotyping and stored until required for somatic cell nuclear transfer.

Transient transfection was performed using Lipofectamine™ 2000 according to the manufacturer’s instructions. Briefly, 3×10⁵ cells were seeded in 6-well tissue culture plates one day prior to transfection. In total, 4 μg of the recombinant constructs per well was transfected into the cells under serum-free DMEM. Following incubation for 4 h, the medium was replaced with DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. A total of 20 h later, various concentrations of dox were treated in transiently transfected cells for an additional 24 h.

Total RNA from MIN6 cells was extracted using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. The concentration of the total RNA was determined by measuring the absorbance at 260 nm. First-strand cDNA was prepared by subjecting total RNA (1 μg) to reverse transcription using Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen Life Technologies) and random primers (9-mers; Takara Bio, Inc.). To determine the optimal conditions for logarithmic phase PCR amplification for target cDNA, aliquots of total cDNA (1 μg) were amplified using different numbers of cycles. The mouse β-actin gene was amplified as the internal control to rule out the possibility of RNA degradation and to control for variations in mRNA concentrations. A linear correlation between PCR product band visibility and the number of amplification cycles was observed for the target mRNA. The mouse β-actin and target genes were quantified using 28 and 30 cycles, respectively. The PCR reactions were denatured at 94°C for 30 sec, annealed at 58°C for 30 sec and extended at 72°C for 30 sec. The PCR products were on a 2.3% agarose gel, stained with ethidium bromide and photographed under UV illumination. The image was scanned using GelDoc EQ (Bio-Rad).

Lysates were prepared in RIPA buffer (50 mM Tris, pH 7.4, 150 mM sodium chloride (NaCl), 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM EDTA and 1 mM PMSF) and protease inhibitor cocktail (Roche Diagnostics). Protein content was determined using the Pierce BCA Micro Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Total proteins obtained by centrifugation (13,000 × g for 20 min at 4°C) were denatured at 95°C for 5 min. A total of 20–50 μg of protein were size-separated by electrophoresis on 13% SDS-polyacrylamide gels and electrophoretically transferred to a nitrocellulose membrane. Non-specific binding was blocked with TBST (20 mM Tris, pH 7.6, 137 mM NaCl and 0.05% Tween-20) containing 5% non-fat milk for 2 h at room temperature (RT). The membrane was probed with the following primary antibodies for 2 h at RT: rabbit polyclonal antibody to Insulin (H-86, sc-9168), diluted 1:1,000; rabbit polyclonal antibody to CREM (X-12, sc-440), diluted 1:1,000 and rabbit polyclonal antibody to β-actin (N-21, sc-130656), diluted 1:1,000. All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The membrane was subsequently exposed to a horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution in TBST) for 1 h at RT.

The unitary tet-on system was composed of an activator cassette and responder cassette (Fig. 1A). The activator cassette had a tTA under the control of the human insulin promoter (−1,432 to +1 nt) which is associated with maximum promoter activity in mouse MIN6 β-cells line according to our previous study (14). The responder cassette contained the porcine ICER Iγ target gene and cDNA encoding ZsGreen1 green fluorescence protein. The expression of both genes was controlled by a TRE promoter. tTA was specifically transcribed in pancreas cells under the control of the human insulin promoter, underwent a conformational change induced by the administration of dox and then bound to the TRE promoter of the responder cassette. Finally, the key gene downstream of the TRE promoter, ICER Iγ, was specifically transcribed in the pancreas cells. Additionally, expression of the green fluorescence marker, ZsGreen1, was observed.

The dox-inducible porcine ICER Iγ vector was linearized and used to transfect miniature pig fibroblasts using a liposomal-mediated gene delivery system. The transfected fibroblasts were selected and maintained with a medium containing G418 (250 μg/ml) for four weeks. Chromosomal integration of the targeting vector was confirmed by a PCR-based method using primer sets specific for the vector (Table I). Genomic DNA extracted from G418-resistant colonies was amplified with primers a and a′ (product size, 992 bp; Fig. 1B) or primers b and b′ (product size, 1,585 bp; Fig. 1C). In total, 12 positive colonies were obtained following two rounds of transfection (Table II). Fibroblasts from the positive colonies may serve as a cellular source for somatic cell nuclear transfer to generate a porcine model that overexpresses ICER Iγ in a dox-inducible, pancreas-specific manner.

To observe tissue-specific expression, we transiently transfected mouse pancreatic β-cells (MIN6) and porcine non-pancreatic fibroblasts. The cells were co-transfected with pCMV-TA and pTRE-ZsGreen1-ICER Iγ constructs as a positive control to confirm the activity of the dox-inducible expression system. In the MIN6 cells expressing pHINSP-TA-TRE-ZsGreen-ICER Iγ, we observed green fluorescence indicative of dox-inducible and pancreas-specific ICER Iγ expression (Fig. 2). In this regard, green fluorescence was also confirmed following dox treatment in a dose-dependent manner (0, 0.1 and 1 μg/ml). However, no fluorescence was observed in either the untreated or dox-treated porcine fibroblasts transiently transfected with pHINSP-TA-TRE-ZsGreen-ICER Iγ due to the pancreas specific insulin promoter (Fig. 3).

To evaluate dox-inducible ICER Iγ expression under the control of the human insulin promoter, reverse transcription (RT)-PCR was performed using mRNA obtained from transiently transfected MIN6 cells and porcine fibroblasts. Consistent to the fluorescence microscopy findings, dox-dose dependent and pancreas specific mRNA expression of ICER Iγ was observed in MIN6 cells (Fig. 4A) following dox treatment but not in porcine fibroblasts (data not presented). Additionally, the expression levels of ICER Iγ in cells transfected with only pCMV-TA or pTRE-ZsGreen1-ICER Iγ were unchanged regardless of the absence or presence of dox. Although ICER Iγ expression in MIN6 cells expressing the pTRE-ZsGreen1-ICER Iγ transgene was observed, the level was consistently low and unaffected regardless of the presence of dox. It should be considered that unknown factors of the transcriptional environment may regulate the gene expression of in vitro systems through introduction of the target gene.

Since the ICER Iγ transgene represses insulin production in vivo (7,8), western blot analysis was performed to determine whether dox-inducible expression of porcine ICER Iγ may inhibit insulin generation. The expression of insulin was evaluated in transiently transfecting MIN6 cells with four combinations of the constructs (Fig. 4B). ICER Iγ protein in cells transfected with only pCMV-TA or pTRE-ZsGreen1-ICER Iγ was not expressed regardless of the absence or presence of dox. Dox-inducible expression of ICER Iγ was observed in cells co-transfected with pCMV-TA and pTRE-ZsGreen1-ICER Iγ, or transfected with pHINSP-TA-TRE-ZsGreen1-ICER Iγ alone. By contrast, insulin expression was decreased in the presence of dox. These findings indicated that dox-inducible ICER Iγ expression decreases insulin expression in mouse pancreas β-cell. Therefore, the extensive induction of ICER Iγ expression may interfere with insulin synthesis and promote the development of type 1 diabetes mellitus. As a result, our dox-inducible ICER Iγ expression system may be a useful tool for studying diabetes mellitus and pre-diabetes in humans by controlling ICER Iγ expression levels through dox administration.