All procedures in this study were carried out in accordance with the Code of Practice for the Care and Use of Animals for Scientific Purposes and approved by the Institutional Animal Care and Use Committee, Dankook University.

Unless otherwise stated, all chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Pig fetuses at 28 to 39 days of gestation were obtained from Minnesota miniature pigs maintained in SPF conditions at Seoul National University. The head, dorsal spine of the medial section, and tail were removed before collection of embryonic fibroblasts. Briefly, small pieces of remaining tissues were washed in Dulbecco’s phosphate-buffered saline (DPBS; Invitrogen, Carlsbad, CA, USA) and minced with a surgical blade in a 100-mm petridish. Cells were then dissociated from the tissues in 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Invitrogen, USA) for 10 min at 39°C. After centrifuging the cell suspension three times at 800×g for 5 min, pellets were resuspended and seeded on 100-mm culture dishes (Corning, Glendale, AZ, USA) and cultured for 6 to 8 days in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1 mM L-glutamine, 100 units/mL penicillin, and 0.5 mg/mL streptomycin in a humidified atmosphere of 5% CO₂ in 95% air. After removal of unattached clumps of cells by washing culture plates with DMEM, attached cells were further cultured until confluent and subcultured at intervals of 5 to 7 days by trypsinization until used for transfection and SCNT.

Fetal fibroblasts were cultured to 80% to 90% confluence on 150-mm plates. After harvested with trypsin and washed with DPBS, cells were transfected using Gene Pulser II (Bio-Rad Laboratories, Hercules, CA, USA). Gene targeting vector, as shown in Figure 1, was introduced into fetal fibroblasts. A total of 2×10⁷ cells were suspended in 400 μL of transfection buffer with 10 μg of linearized targeting vector. Electroporation was performed at 250 V, 1,000 mF. Starting from 24 h after transfection, genetically modified cells were selected with culture medium containing 500 μg/mL of G418 (Invitrogen, USA) for 10 to 14 days. After antibiotic selection, G418-resistant colonies were isolated and transferred onto 0.1% gelatin-coated dish for clonal culture. Cells were continuously cultured at 39°C in a humidified atmosphere containing 5% CO₂ and 95% air. Genomic DNA was extracted from cells of G418-resistent colonies, and αGT gene targeting events were screened by polymerase chain reaction (PCR) using specific primer sets described previously [2].

The in vitro maturation (IVM) of oocytes and NT were performed as described previously [2]. Briefly, 42 h after the onset of IVM, oocytes were enucleated with a 20-μm (internal diameter) glass pipette by aspiration of the first polar body and the second metaphase plate with a small volume of surrounding cytoplasm in HEPES-buffered TCM-199 supplemented with 0.3% bovine serum albumin (BSA) and 5 μg/mL cytochalasin B. After enucleation, oocytes were stained with 5 μg/mL bisbenzimide (Hoechst 33342) for 5 min and observed under a Nikon TE-300 inverted microscope equipped with epifluorescence. Oocytes containing DNA materials were excluded from the subsequent experiments. Fibroblasts were trypsinized into single cells and transferred into the perivitelline space of enucleated oocytes. The resulting couplets were equilibrated for 1 min in 0.3 M mannitol solution containing 0.5 mM HEPES, 0.05 mM CaCl₂, and 0.1 mM MgCl₂ in a chamber containing two electrodes. Using a BTX Electro-Cell Manipulator 2001 (Harvard Apparatus, Holliston, MA, USA), couplets were fused with a double DC pulse of 1.5 KV/cm for 45 μs. Following electrical stimulation, reconstructed oocytes were cultured in porcine zygote medium-3 supplemented with 3 mg/mL fatty acid-free BSA and 5 μg/mL cytochalasin B for 3 h in order to suppress extrusion of the second polar body.

On the next day of SCNT, reconstructed embryos were surgically transferred to naturally cycling surrogate sows on the second day of standing estrus. Abdominal ultrasonograpy to test for pregnancy was performed at days 25 to 30 after the embryo transfer. Thereafter, pregnant recipient were examined by ultrasound weekly. At near full-term birth, fetal heartbeat or movement was monitored by Doppler ultrasonography.

Fibroblasts were cultured from ear skin biopsies of the heterozygous αGT KO piglets as previously reported [2]. Selection of homozygous αGT KO cells derived from LOH was performed as described elsewhere [11,12]. Briefly, 5×10⁵ heterozygous αGT KO cells were washed twice with DPBS, resuspended with 1 mL of DPBS containing 4 μg biotin-conjugated IB4-lectin (EY Laboratories, San Mateo, CA, USA) in a 1.5-mL tube, and incubated for 1 h on ice with tapping the tube to prevent precipitation of the cells. After washing cells twice with DPBS by centrifugation, 4 mg of Dynabeads M-280 Streptavidin (Invitrogen, USA) was added to the pellet and the resulting mixture was again incubated as same as the IB4-lectin treatment. Then, the tube was placed on a magnet for 2 to 3 min, and the supernatant was transferred to a new tube on a magnet. This procedure was repeated three times. The final supernatant was cultured in DMEM containing 10% (v/v) FBS in 60-mm culture dish for 10 to 14 days until colonies reached approximately 2 mm in diameter. The culture dish containing fibroblast colonies was washed twice with DPBS and covered with dimethylpolysiloxane (DMPS). Colonies were dissociated by injecting 30 μL of 0.25% trypsin-EDTA on top of each colony underneath DMPS using micropipette. After incubation for 5 min at 39°C, fibroblasts dissociated from each colony were transferred onto 0.1% gelatin-coated 24-well culture dish for clonal culture. Clones of homozygous αGT KO were analyzed by PCR as previously reported [2]. Briefly, genomic DNA was prepared from fibroblasts using DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Using Maxime PCR Premix Kit (Intron Biotechnology, Seongnam, Korea) in 20 μL reaction volume, amplification of the target gene was carried out using forward 5′-AGAGGTCGTGACCATAACCAGAT-3′ and reverse 5′-AGCCCATGCTGAACATCAAGTC-3′ primers. Conditions for PCR were as follows: 30 cycles of 15 s at 94°C, 30 s at 65°C, 10 min+20 s increase/cycle at 68°C; and one final cycle of 7 min at 68°C. Amplification products (7.4 and 9.2 kb as shown in Figure 1) were analyzed by 0.4% agarose gel electrophoresis. Colonies that LOH was confirmed by PCR were used for SCNT to produce homozygous αGT KO pigs.

Karyotype analysis of homozygous αGT KO fibroblasts was performed by GenDix, Inc (Seoul, Korea), a commercial provider of karyotyping. Briefly, 200 μL of colecemid (Invitrogen, USA) was added to cells and incubated overnight at 39°C in 5% CO₂. Cells were trypsinized, harvested, and centrifuged for 5 min at 400×g. After 10 μL of hypotonic solution (0.075 M KCl) was added to the cell pellet, cells were resuspended, incubated for 20 min at 39°C in 5% CO₂, and centrifuged for 5 min at 400×g. Then, hypertonic solution was removed, and 500 μL Carnoy’s fixative (methanol:acetic acid = 3:1) was added and mixed with the cells. After fixative containing fibroblasts was dropped and spread on clean glass slides, the slides were baked 60°C for 30 min and treated with 50% H₂O₂ for 3 min. Finally, followed by karyotypic analysis following Giemsa staining (GTG banding) was performed using ChIPS-Karyo (Chromosome Image Processing System, GenDix, Seoul, Korea).

To evaluate the expression levels of αGal epitope on cell surface, fibroblasts obtained from wild type, heterozygous αGT KO, and homozygous αGT KO pigs were analyzed by flow cytometry. Cultured cells were harvested, washed with DPBS and resuspended at 7×10⁴/mL. Then cells were labeled with fluorescein isothiocyanate (FITC)-conjugated IB4 and analyzed using BD FACSCalibur Cell Analyzer (BD Biosciences, San Jose, CA, USA).

Southern blot hybridization was performed using DIG High Prime DNA Labeling and Detection Kit 2 (Roche Molecular Systems, Pleasanton, CA, USA). Ten micrograms of genomic DNA were digested with a Bst EII (Takara, Shiga, Japan) overnight at 60°C. As represented in Figure 1, the digestion generates a 6.8 kb fragment in the wild allele and an 8.1 kb fragment in the targeted allele. Samples were separated on a 0.8% agarose gel. Following electrophoresis, restricted genomic DNA was transferred to a nylon membrane (Roche, Switzerland). Then, the membrane was hybridized with a 520 bp DIG-labeled probe in DIG Easy Hyb solution (Roche Molecular Systems, USA) for 16 h at 53°C. After the hybridization, membrane was washed twice in 0.5× SSC with 0.1% sodium dodecyl sulfate at 68°C and exposed to a camera for 10 min.

A total of 2×10⁷ fetal fibroblasts were transfected using αGT gene-targeting vector, and 87 colonies were obtained after antibiotic selection. Among these colonies, two (2.3% efficiency) were identified as heterozygote αGT KO. One of the two was used for subsequent SCNT experiment. A total of 559 cloned embryos were produced by SCNT and transferred to five surrogates. Two pregnancies went to term, leading to delivery of three female piglets by cesarean section (Figure 2A). Genomic DNA was extracted from tail tip of piglets and analyzed by PCR. All of three piglets were confirmed to be heterozygote αGT KO (Figure 2B).

After IB4-Dynabead selection, 22 colonies were isolated from 5×10⁵ cultured fibroblasts obtained from heterozygous αGT KO cloned piglets. Based on PCR analysis, 15 colonies were confirmed to have LOH with biallelic disruption of αGT presumably by mitotic recombination (68.2% LOH efficiency). One of the colonies was chosen for expansion of cells by subculture and subjected to karyotypic analysis. Subcultured homozygous αGT KO fibroblasts demonstrated normal porcine karyotype (Figure 3) and subsequently used for SCNT.

Using the same procedure for the production heterozygous αGT KO pigs, a total of 729 cloned embryos were produced and transferred to four surrogates. Two pregnancies went to term, leading to delivery of four piglets with three viable and one stillborn by cesarean section (Figure 4A). The stillborn piglets had no gross clinical and anatomical abnormalities, and the cause of death was not clear. As represented in Figure 4B and 4C, genomic DNA analyses by PCR and Southern blot showed that all of the piglets were homozygotes with disruption in both alleles of αGT gene.

To examine the expression of αGal epitopes on cell membrane, ear skin fibroblasts obtained from wild type, heterozygous, and homozygous αGT KO piglets were reacted with FITC-conjugated IB4 lectin. As shown in Figure 5, unlike wild type and heterozygous αGT KO cells, no fluorescence was observed from homozygous αGT KO fibroblasts, suggesting αGal antigen was not expressed on cell surface of homozygous αGT KO fibroblasts.