Mouse embryonic 3T3-L1 fibroblast adipose-like cells were purchased from the Korean Cell Line Bank (Seoul, South Korea). 3T3-L1 preadipocytes were incubated, unless otherwise indicated, at 37°C and, unless otherwise indicated, all cell culture materials were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). On day 30 of pregnancy, porcine fibroblasts were obtained from a miniature pig fetus (Yucatan pig; Optifarm Solution Inc., Gyeonggi-do, South Korea). Fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS; WelGENE, Daegu, South Korea), 50 U/ml penicillin and 50 μg/ml streptomycin in a humidified 5% CO₂ atmosphere.

Genomic DNA was isolated from the fibroblasts using a G-DEX™ IIc genomic DNA extraction kit (Intron Biotechnology, Seoul, South Korea). All experimental procedures and animal use were approved by the Ethics Committee of the Chungbuk National University. Genomic DNA (1 μg) was amplified in a 20 μl PCR containing 1 unit LA-Taq polymerase (Takara Bio, Inc., Shiga, Japan) for long-range PCR or 1 unit i-star Taq polymerase (Intron Biotechnology), 2 mM dNTPs (Takara Bio, Inc.) and 10 pmol each specific primer. All primers are presented in Table I. PCR conditions were as follows: denaturation at 95°C for 30 sec, annealing at 62°C for 30 sec and extension at 72°C for 1 or 2 min. PCR products were separated on a 0.7% agarose gel, stained with ethidium bromide, imaged under UV illumination and processed for cloning. The gel image was scanned using Gel Doc EQ (Bio-Rad, Hercules, CA, USA).

Total RNA was extracted from 3T3-L1 cells or pig liver (Yucatan pig; Optifarm Solution Inc.) using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. Total RNA concentration was determined by measuring absorbance at 260 nm (Epoch microplate spectrophotometer; BioTek, Winooski, VT, USA). First strand cDNA was prepared by subjecting total RNA (1 μg) to reverse transcription using M-MLV reverse transcriptase (Invitrogen Life Technologies) and random primers (9-mers; Takara Bio Inc.). Optimal conditions for logarithmic phase PCR amplification of the target cDNA were determined by amplifying aliquots of total cDNA (1 μg) using a different number of cycles. The cytochrome c oxidase subunit 1 (1A) gene was used as an internal control to eliminate the possibility of RNA degradation and to account for variation in mRNA concentration. A linear correlation between the PCR product band visibility and the number of amplification cycles was observed in the target mRNA products. The 1A gene and target gene, insulin, were quantified using 28 and 30 cycles, respectively. PCR conditions were as follows: denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec and extension at 72°C for 30 sec. PCR products were separated in a 2.3% agarose gel, stained with ethidium bromide and imaged under UV illumination. The image was scanned and band densities were analyzed using Gel Doc EQ (Bio-Rad).

Restriction enzymes were obtained from Takara Bio, Inc. Specific regions of the aP2 promoter were prepared by long-range PCR using porcine genomic DNA as a template and specific primers containing restriction enzyme sites (MluI at 5′ end or BglII at 3′ end). Amplified fragments were digested with MluI and BglII and ligated into the promoterless pGL3-Basic luciferase expression plasmid (Promega Corporation, Madison, WI, USA). The 11β-HSD1 expression cassette plasmid was produced in several steps. 11β-HSD1 cDNA was prepared by PCR using cDNA from pig liver as a template. The amplified fragments were inserted into the recombinant pGL3 construct containing the pig aP2 promoter region (−2,826 to +51 nt). For the selection cassettes, the enhanced green fluorescent protein (EGFP) gene was amplified from the pIRES2-EGFP plasmid (Clontech Laboratories Inc., Mountain View, CA, USA), digested with EcoRV and BamHI and inserted into the pIRESneo plasmid (Clontech Laboratories Inc.). The EGFP and neomycin-resistant (Neor) genes were amplified by PCR, digested with SalI and ligated into the recombinant pGL3 vector encoding pig 11β-HSD1 cDNA controlled by the porcine aP2 promoter. Finally, sequences of the targeting vector were confirmed by nucleotide sequencing (Genotech Corporation, Daejeon, South Korea).

Transient transfection was performed using Lipofectamine™ 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. To account for varied transfection efficiencies of the various luciferase constructs, the Rous sarcoma virus (RSV)-lacZ plasmid was co-transfected with the luciferase constructs containing the pig aP2 promoter, as previously described (22,23). Briefly, 3×10⁵ cells were seeded in 6-well tissue culture plates (Invitrogen Life Technologies) 1 d prior to transfection. Constructs (4 μg) containing the aP2 promoter and RSV-lacZ plasmid (0.5 μg) were co-transfected into the cells with DMEM. Following an incubation period of 4 h, the media was replaced with DMEM containing 10% FBS and glucose (0.1 or 4 mM) and the cells were incubated for an additional 48 h at 37°C. Cell lysates were assayed for luciferase activity using a luciferase assay system (Promega Corporation). Luminescence was measured using a GloMax 20/20 Luminometer (Promega Corporation). β-galactosidase activity was measured using a β-galactosidase enzyme assay system (Promega Corporation). Relative luciferase activity was calculated as luciferase activity/β-galactosidase activity (%).

Porcine fibroblasts were transfected with the linearized targeting vector using Lipofectamine 2000. Following 24 h transfection, the medium was replaced with DMEM supplemented with 10% FBS and 250 μg/ml G-418 (Roche Diagnostics, Indianapolis, IN, USA) for 4 weeks. Antibiotic-resistant colonies were further selected according to EGFP expression observed with a fluorescence microscope (Nikon, Tokyo, Japan). Antibiotic and visually selected colonies were subjected to PCR-based genotyping and stored until required for SCNT.

Data are presented as the mean ± SEM. A statistical analysis was performed by Student’s t-test for two-pair comparisons. P<0.05 was considered to indicate a statistically significant difference.

Prior to the generation of the 11β-HSD1 cDNA construct, a pig aP2 promoter that was hypothesized to be activated in adipocytes was produced. The aP2 gene is typically expressed in adipose tissue and the maximum promoter activity of aP2 in the adipose tissue was previously confirmed (24). A series of pig aP2 gene promoters with sequential deletions were inserted into the luciferase (Luc) gene in a promoterless pGL3-basic vector. The aP2 promoter constructs were introduced into the mouse embryonic fibroblast-adipose 3T3-L1 cells and the promoter activities were evaluated according to Luc expression. As presented in Fig. 1, the promoter region between −2,826 and +15 nt resulted in an ~20-fold increase of Luc activity compared with the promoterless construct. These observations indicate that −2,826 to +15 nt of the aP2 promoter was the best candidate adipocyte-specific expression promoter.

Following verification that the aP2 promoter construct containing −2,826 to +15 nt had maximal activity, the construct was subcloned with porcine 11β-HSD1 cDNA. The resulting aP2/11β-HSD1 construct was capable of expressing 11β-HSD1 mRNA in an adipocyte-specific manner. 11β-HSD1 mRNA levels increased in the transiently-transfected 3T3-L1 cells (Fig. 2). These observations indicate that the recombinant aP2/11β-HSD1 construct was appropriate for expressing pig 11β-HSD1 in an adipose tissue-specific manner.

Our constructs were composed of two parts, an 11β-HSD1 expression cassette and a selection cassette (Fig. 3A). The 11β-HSD1 expression cassette contained pig 11β-HSD1 cDNA with the porcine aP2 promoter region (−2,826 to +15 nt). The selection cassette contained EGFP and Neor genes, whose expression was regulated by a cytomegalic virus promoter. Expression of the EGFP and Neor genes enabled selection of the cells of interest using antibiotics and visual screening.

The targeting construct was linearized and used to transfect porcine fibroblasts isolated from Yucatan pigs. Fibroblasts were incubated in medium containing G-418 (250 μg/ml) for 4 weeks to select for the stable transfectants. Identification of Neor colonies as transgenic fibroblasts was confirmed by EGFP expression (Fig. 4). The clones were further identified by PCR-based methods using primers specific for the targeting constructs. Genomic DNA from clones expressing porcine 11β-HSD1 was analyzed with an a-b primer set (amplicon size, 740 bp; Fig. 3B) or with c-d primers (amplicon size, 1 kb; Fig. 3C). Transfection efficiency was ~89% (Table II). These observations indicate that the cell model may be useful for establishing a transgenic fibroblast system for creating porcine metabolic syndrome models by expressing adipose tissue-specific 11β-HSD1.