The female mixed breed dogs (1- and 7-year-old, 20–25 kg) used for our study were housed singly. They were fed a standard commercial dog diet (Suwon Purina, Suwon, Kyunggi-Do, Korea) once a day and given water ad libitum in accordance with the animal study guidelines of the SooAm Biotech Research Foundation for Accreditation for Laboratory Animal Care.

Unless otherwise indicated, all cells were grown at 37°C. All cell culture materials were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA) and the other chemicals from Sigma-Aldrich (St. Louis, MO, USA). Adult fibroblasts were isolated from the abdominal skin of a 2-year-old beagle dog (Medicare Animal Hospital, Suwon, Kyunggi-Do, Korea). Normal or cloned fetal fibroblasts were from fetuses recovered on gestation Day 25 after mating or embryo transfer. Fetuses of the beagles were retrieved aseptically by laparotomy. The isolated tissues were minced with a surgical blade in a culture dish (35- or 100-mm; Becton Dickinson, Lincoln Park, NJ, USA) and dissociated in DMEM supplemented with 0.1% (w/v) trypsin/1 mM EDTA for 1–2 h. Trypsinized cells were subsequently cultured for 6–8 days in DMEM supplemented with 10% (v/v) FBS, 1 mM sodium pyruvate, 1% (v/v) non-essential amino acids (NEAA) and a 10 μg/ml penicillin/streptomycin solution. When the cells were fully confluent, they were collected by trypsinization and were frozen in DMEM supplemented with 40% FBS and 10% dimethyl sulfoxide (DMSO).

Genomic DNA was isolated with a G-DEX™ IIc Genomic DNA extraction kit (Intron Biotechnology, Suwon, Korea). Genomic DNA (1 μg) was amplified in a 20-μl PCR reaction containing 1U Ex-Taq polymerase (Takara Bio, Inc., Shiga, Japan), 2 mM dNTP (Takara Bio, Inc.) and 10 pmol of specific primers. The details of all primers are provided in Table I. The PCR reactions were denatured at 95°C for 30 sec, annealed at 62°C for 30 sec and extended at 72°C for 1 or 3 min. The PCR products (8 μl) were separated on a 0.7 % agarose gel, stained with ethidium bromide and photographed using Gel Doc EQ (Bio-Rad, Hercules, CA, USA).

Unless otherwise indicated, all restriction enzymes were obtained from Takara Bio, Inc. The canine PEPCK promoter from −3,180 nucleotide (nt) to +1 nt (+1, the transcriptional start site) was isolated by PCR using genomic DNA from beagle fibroblasts as a template and included an NheI site at the 5′ end and a NcoI site at the 3′ end. Amplified fragments were digested by the restriction enzymes and ligated into the promoterless pGL3-Basic vector (Promega Corporation, Madison, WI, USA). The PEPCK promoter-conjugated luciferase construct shown in Fig. 1, was transiently transfected into hepatoma cells (H4IIE; American Type Culture Collection, Manassas, VA, USA) using Lipofectamine™ 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. To control different transfection efficiencies of the various luciferase constructs, the Rous sarcoma virus (RSV)-lacZ plasmid was co-transfected into H4IIE cells.

Briefly, 3×10⁵ cells were seeded in 6-well tissue culture plates (BD Biosciences, Franklin Lakes, NJ, USA) one day before transfection. Four micrograms of the luciferase constructs and 0.5 μg of RSV-lacZ plasmid were co-transfected into the cells. After transfection for 4 h, media were replaced with DMEM containing 10 % FBS and 1 mM dexamethasone for other 48 h. Cellular lysates were prepared using 150 μl of reporter lysis buffer (Promega Corporation) and assayed for luciferase activity using the luciferase assay system (Promega Corporation). Luminescence was measured using a GloMax^® 20/20 Luminometer (Promega Corporation) and β-galactosidase activity was measured using a β-galactosidase enzyme assay system (Promega Corporation). Promoter activity was calculated as relative luciferase normalized by β-galactosidase activity (%).

PEPCK cDNA was prepared by PCR using cDNA from beagle liver as a template. Amplified fragments were digested with NcoI and XbaI and ligated into the recombinant pGL3_PEPCK promoter (−746 to +1 nt) plasmid to replace the luciferase gene. The selection cassette plasmid was made in several steps. Enhanced green fluorescence protein (EGFP) was amplified by PCR using pIRES2_EGFP (BD Biosciences) as a template. The fragments were digested with EcoRV and BamHI and ligated into the pIRES_Neo vector (BD Biosciences). The selection cassette was amplified by PCR using the recombinant pIRES_EGFP plasmid containing a neomycin resistance gene (Neo^r). The selection cassette fragments were digested with MluI and NheI, and ligated into the recombinant pGL3_PEPCK promoter_PCPEK cDNA plasmid. The targeting vectors sequences were confirmed by nucleotide sequencing (Genotech Co., Ltd., Daejeon, Korea).

The structure of the targeting vector is shown in Fig. 3A. The selection cassette construct contained the cytomegalovirus (CMV) promoter, EGFP gene and Neo^r. The expression cassette consisted of the dog PEPCK promoter region (−746 to +1 nt), dog PEPCK cDNA and a simian virus (SV) 40 poly-A tail signal. The targeting vector was linearized by MluI digestion and transfected into normal fetal fibroblasts by Lipofectamine 2000 (Invitrogen Life Technologies).

Normal fetal fibroblasts (2×10⁵) at passage 2 were seeded in a 35-mm culture plate (BD Biosciences) 1 day before transfection. Cells were transfected with 4 μg of linearized targeting vector and then cultured in DMEM containing 10% FBS and a high dose of antibiotics (600 μg/ml of G418; Sigma) for 20 days. The concentration of antibiotics was further reduced to 300 μg/ml for additional days until single colonies appeared. The neomycin-resistant colonies were reseeded in 24-well plates (BD Biosciences) to expand the colonies for further analysis. Following several passages, the transgenic cells screened for EGFP expression and chromosomal integration of targeting vector was further confirmed by PCR-based genotyping. The colonies were expanded and frozen (6×10⁵ cells/aliquot) prior to their subsequent use for SCNT.

Estrous of the female dogs was followed weekly by observing vulval bleeding to detect the onset of the heat period. During heat, a blood sample (2 ml) was collected daily at the same time by cephalic venipuncture and serum progesterone levels were assayed using a Cobas E411 (Roche Diagnostics, Indianapolis, IN, USA). Oocyte retrieval and cloned embryo transfer were performed using general surgical procedures as described in our previous study (13). Briefly, the oviducts were flushed upward with TCM-199 (Invitrogen Life Technologies) from oocyte donor dogs under surgical procedures. The oocytes were denuded and washed for nuclear transfer. The cells were then stained with 5 μg/ml bisbenzimide and mounted. Oocytes with a nucleus and extruded first polar body (metaphase II) were counted and used for a routine nuclear transfer procedure.

After retrieval, metaphase II oocytes were stripped from cumulus cells and enucleated by squeezing out the first polar body and the metaphase II plate into a small amount of surrounding cytoplasm using a glass pipette. The cells were transferred to the perivitelline space of the enulceated oocytes. Using a fine pipette, a trypsinized fetal fibroblast with a smooth cell surface was transferred to the perivitelline space of an enucleated oocyte. The couplets were equilibrated in a 0.26 M mannitol solution containing 0.5 mM of HEPES, 0.1 mM of CaCl₂ (Sigma) and MgSO₄ (Sigma) for 4 min and then transferred to a chamber containing two electrodes overlaid with a mannitol solution (Sigma). The couplets were fused with two direct current pulses of 1.9–2.2 kV/cm for 30 μsec using a BTX Electro-Cell Manipulator 2001 (BTX, Inc., San Diego, CA, USA). Only the fused embryos were selected and cultured for 2 h in modified synthetic oviductal fluid (mSOF). The formula of the mSOF was basically the same as the original formulation (11) except of the concentration of glucose (1.5 mM) and the addition of 2% essential MEM, 1% nonessential amino acids, 8 mg/ml of BSA and 1% (v/v) insulin, transferrin and selenium (Invitrogen Life Technologies).

Embryo recipients were anaesthetized as previously described for oocyte retrieval and placed in a ventral recumbent position. The ovary with a greater number of corpus lutuem was approached for ventral laparotomy. The fat layer covering the ovary was gently grasped with forceps and suspended with a suture to exteriorize the fimbriated end of the oviduct. The embryos were loaded into a tomcat catheter (Sovereign, Sherwood, MO, USA) and gently transferred to the 2/3 distal position of the oviduct without insufflating air. Surrogates were checked for pregnancy by transabdominal ultrasound 25–30 days after embryo transfer.

Parentage analysis was performed for the puppies derived from SCNT to confirm the identity of the donor cell used for nuclear transfer. Genomic DNA was extracted from each newborn puppy, recipients and donor cells. DNA microsatellite markers (PEZ1, PEZ3, PEZ5, PEZ6, PEZ8, PEZ12, PEZ20, FHC 2010, FHC 2054 and FHC 2079) were used to confirm the genetic identity of the cloned puppies, the fetuses and fibroblasts used as donor cells.

Total-RNA was extracted from the liver biopsy tissues of the cloned and transgenic dogs using TRIzol reagent (Invitrogen Life Technologies) according to the protocol provided by the manufacturer and the concentration of total-RNA was determined by measuring the absorbance at 260 nm. First strand complementary DNA (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.). Quantitative real-time PCR was performed with 1 μl of cDNA template added to 10 μl of 2X SYBR Premix Ex Taq (Takara Bio, Inc.) containing 10 pmol each of the specific primers. Reactions were carried out for 40 cycles. The cycling parameters were as follows: denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec and extension at 72°C for 15 sec. Fluorescence intensity was measured at the end of the extension phase of each cycle. The threshold value of the fluorescence intensity for all the samples was set manually. The reaction cycle at which the PCR products exceeded this fluorescence intensity threshold in the exponential phase of amplification was designated as the threshold cycle (Ct).

Expression of the target gene was quantified relative to the level of an internal control gene (GAPDH) based on the comparison of Cts at a constant fluorescence intensity. The amount of transcript present was inversely related to the observed Ct. For every two-fold dilution in the amount of transcript, the Ct was expected to increase by 1. Relative expression (R) was calculated using the equation R=2^{−[ΔCT sample−ΔCT control]}. To determine a normalized arbitrary value for each gene, every data point was normalized to the GAPDH control gene as well as to their respective controls.

Liver biopsy tissues were harvested, washed twice with ice-cold PBS and resuspended in 20 mM Tris-HCl buffer (pH 7.4) containing protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml pepstatin A and 1 μg/ml chymostatin) and phosphatase inhibitors (5 mM Na₃VO₄ and 5 mM NaF). All inhibitors were obtained from Sigma. Whole cell lysates were prepared with 20 strokes of a Dounce homogenizer followed by centrifugation at 13,000 × g for 20 min at 4°C. Protein concentrations were determined using a BCA assay (Sigma). The proteins (50 μg) were separated with 12% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membranes were probed with antibodies against PEPCK (diluted 1:1,000; Cayman Chemical, Ann Arbor, MI, USA) or β-actin (1:4,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Horseradish peroxidase-conjugated anti-rabbit IgG (diluted 1:2,000; Santa Cruz Biotechnology, Inc.) were used to detect primary antibody binding and was visualized by exposure to X-ray film (Bio-Rad). The band intensities were quantified with the Gel Doc EQ system (Bio-Rad).

Data analysis was conducted using SPSS for Windows (version 15; SPSS Inc., Chicago, IL, USA) and a graph was prepared with GraphPad Prism (version 4.0) software (GraphPad Software, Inc., San Diego, CA, USA). Data for the pregnancy rate were compared using an ANOVA and the results from western blot analysis were compared using a t-test with post hoc analysis. P-values <0.05 were considered to be significant.

To determine the promoter region of the canine PEPCK gene which has the maximum activity for tissue-specific expression, we generated a series of promoter variants containing different deletions of the upstream human insulin promoter region (−3,180 to +1 nt) using beagle genomic DNA. The fragments were linked to the luciferase (Luc) gene in a promoterless pGL3-Basic vector. These PEPCK promoter constructs were finally introduced into the H4IIE hepatoma cell line and the relative promoter activities were measured in the presence of dexamethasone (Dex). As shown in Fig. 1A, pGL3-2Kb (−2,349 to +1 nt) and pGL3-0.7Kb (−746 to +1 nt) induced 110- and 130-fold increases in luciferase activity, respectively. However, a more modest increase in luciferase activity was observed with pGL3-3Kb (−3,180 to +1 nt) and pGL3-1Kb (−1,018 to +1 nt). Thus, we identified the two promoter regions, pGL3-2Kb and pGL3-0.7Kb, associated with the greatest luciferase activity.

We further investigated whether these selected promoters contributed to PEPCK transcription and translation. PEPCK cDNA was replaced with Luc genes in pGL3-2Kb and pGL3-0.7Kb to generate pGL3-2Kb-PEPCK and pGL3-0.7Kb-PEPCK. These constructs were introduced into H4IIE cells. The protein levels of PEPCK were higher for pGL3-0.7Kb-PEPCK than pGL3-2Kb-PEPCK (Fig. 1B). Thus, pGL3-0.7Kb-PEPCK was selected as the expression cassette for the targeting vector containing dual selection marker genes, EGFP and Neo^r (Fig. 1C).

To compare the in vivo developmental competence among different SCNT nuclear sources, fibroblasts from adult dogs, normal fetuses, or cloned fetuses were introduced into oocytes as donor cells for nuclear transfer and sequentially implanted into recipients. The fusion, pregnancy, and delivery rates were further measured (Table II). For the adult fibroblasts, 134 oocytes were used for nuclear transfer and 103 (76.9%) reconstructed embryos were transferred to 6 recipients. For the cloned fetal fibroblasts, 118 oocytes were used for nuclear transfer and 115 (97.5%) reconstructed embryos were transferred to 10 recipients. For the fetal fibroblast, 66 oocytes were used for nuclear transfer and 51 (77.3%) reconstructed embryos were transferred to 4 recipients. The highest fusion rates before embryo transfer were observed for the cloned fetal fibroblast group (P<0.05) comparing to the other cells. For the adult fibroblasts, one pregnancy was identified at mid-term gestation (16.6%) and reached full-term (one offspring). For the cloned fetal fibroblasts, two pregnancies were identified at mid-term (20.0%), one pregnancy reached full-term and the other was aborted on Day 45. For the fetal fibroblasts, two recipients were confirmed pregnant and reached full-term (one singleton and one set of twins). Pregnancy rates on Day 30 were not significantly different among the cell sources. However, fetal fibroblasts as a source for nuclear transfer resulted in the best cloning efficiency (5.9%) comparing to adult (1.0%) or cloned fetal fibroblasts (0.9%).

Normal fetal fibroblasts, nuclear donor cells, were introduced by the targeting vector. After screening with antibiotics, the identity of positive clones was confirmed with PCR-based genotyping. The primers a and b were used to demonstrate integration of the selection cassette (Fig. 2A). Chromosomal insertion of the expression cassette was confirmed by the primers c and d (Fig. 2B). Endogenous PEPCK gene generated a 5.2-kb band containing intron regions whereas the inserted expression cassette produced a 2.7-kb band. The cells found to express EGFP and possessing resistance against neomycin, were subjected to nuclear transfer.

The results of embryo transfers are summarized in Table III. A total of 75 oocytes were from 7 egg donors (10.7 oocytes/egg donor) and subjected to nuclear transfer using the transgenic cells. Forty-seven embryos that had successfully fused (72.3%) were transferred into 5 recipients and 2 surrogates (S2 and S3) delivered 3 puppies (Table IV). One (DM-1) of the puppies from S2 was healthy but the other (DM-2) died within 24 h. This scenario was also observed in our previously reported study (22). Although DM-3 from S3 was born by Cesarean section, it was healthy with a normal body weight (360 g). The two surviving puppies (DM-1 and DM-3) expressed EGFP in their claws and skin (Fig. 3A), and primary cells from these puppies were further found to express EGFP when examined under a microscope (Fig. 3B). Genetic identities of the cloned puppies with the nuclear donor fibroblasts were confirmed by microsatellite parentage analysis (Fig. 3C). Thus, two puppies were visually confirmed to be transgenic dogs by EGFP expression and further identified as cloned dogs by a microsatellite assay.

To confirm chromosomal insertion of the targeting vector, genomic DNA of 3 puppies (DM-1, DM-3 and control) were prepared from their umbilical cords and PCR-based geno-typing was performed. DM-1 and DM-3 had the PEPCK expression cassette (2.7 kb) and the endogenous PEPCK gene (5.2 kb) but the age-matched control had only the endogenous gene (Fig. 4A). Integration of the selection cassette was further confirmed by PCR (Fig. 4B). In addition, functional activity of the PEPCK gene in the targeting vector was estimated by real-time PCR (Fig. 4C) and immunoblotting analysis of a liver biopsy sample from DM-3 (Fig. 4D). In DM-3, PEPCK mRNA expression was 2 times higher than that of the wild-type control and PEPCK protein expression was induced. Although we confirmed that DM-3 overexpressed the PEPCK gene, this animal has not presented any symptoms associated with type 2 diabetes mellitus thus far. We continue to monitor DM-3 for the onset of diabetes-like symptoms because this type of diabetes is a late-onset disease.