Protocols for the use of animals in this study were approved by the Animal Care Committee of the National Institute of Agrobiological Sciences.

Construction of Vectors

The transgene, termed pEGFPac (Fig. 1), was constructed by inserting CAG-EGFP fragment into pPGK-pac-p(A) cassettes. Briefly, pCAG-EGFP, an EGFP-expression cassette, was first constructed according to Okabe et al.. The EGFP cDNA was PCR amplified from pEGFP-C1 (Clonetech, Palo Alto, CA) using F1 (5′-ttgaattcgccaccatggtgagc-3′) and R1 (5′-ttgaattcttacttgtacagctcgtcc-3′) primers. After digestion of the amplified cDNA fragments with EcoRI, these fragments were subcloned into the EcoRI site of the pCAGGS expression vector that contains the cytomegalovirus enhancer, chicken pactin promoter, and a portion of second intron, third exon, and 3′ noncoding region of the rabbit pglobin gene. The PGK-pac-p(A) cassette obtained from digesting pPGK-pac-p(A) with SalI, was introduced into the PstI site of pCAG-EGFP by blunt-end ligation. The resulting plasmid was termed pEGFPac, and sequenced to check that the insert had been properly inserted. A 4.8-kilobase (kb) fragment containing EGFPac was isolated, before gene transfer, from the SalI-digested pEGFPac after electrophoresis on a 0.8% agarose gel.

Isolation of Fetal Somatic Cells

A pregnant female sow (Landrace) was killed at 73 days, and fetuses were dissected out. The associated yolk sac and amnion were removed from the fetuses under germ-free conditions. After washing with Dulbecco modified phosphate buffered saline without Ca2+ and Mg2+ [PBS], two fetuses were minced using scissors and dispersed by incubating them at 37°C for 20 min in 10 ml of PBS containing 1% (w/v) of collagenase (C9407; Sigma Co. Ltd., St. Louis, MO) and 0.1% (w/v) of trypsin (159090-46; Gibco, Grand Island, NY). Dissociated somatic cells were then collected by low-speed centrifugation at 4°C and then resuspended in PBS. The number of viable cells was counted using the improved Neubauer hemocytometer (Kayagaki, Tokyo, Japan) after staining with trypan blue. A small portion of cells was subjected to a puromycin sensitivity assay as described below. The remaining cells were immediately subjected to electroporation-mediated gene transfer as described below, and the remaining cells were deep frozen using culture medium containing 10% (v/v) dimethyl sulfoxide (DMSO) (D2650; Sigma Co. Ltd.).

Puromycin Sensitivity Assay

Stock solution (10 mg/ml) of puromycin (P8833; Sigma Co. Ltd.) was prepared by dissolving puromycin in distilled water at the appropriate concentration and stored at 4°C. Media containing variable amounts of puromycin were freshly prepared by adding the appropriate volume of puromycin stock solution.

To determine the optimal concentration of puromycin for selecting EGFPac-transfected cells, a puromycin resistance test was performed with fetal porcine somatic cells. Cells were seeded in 24-well plates at a density of 2.5 X 104 cells per well and cultured in medium containing 0.5-6 |xg/ ml puromycin for 7 days. A total of 12 wells containing puromycin at indicated concentrations were used per assay. This assay was repeated three times. Cell viability was examined by using the Cell Titer-Glo Luminescent Cell Viability Assay Kit (G7571; Promega Co., Madison, WI). Cells from 12 independent wells were separately measured for luciferase activity. Luciferase activity was measured using a luminometer (CT-9000D; Dia-Iatron, Tokyo, Japan) following the protocols described by the manufacturer. Because there is a possibility that each well might contain a variable number of cells, data on luciferase activity were normalized against the protein concentration in each well. The resulting data on 12 independent cells were statistically analyzed using a two-tailed Student t-test. Based on these treatments, cell viability was plotted and analyzed.

The survivability of primary-cultured fetal porcine somatic cells was also examined by culturing in a medium containing 2 |xg/ml of puromycin for up to 7 days. Cells were harvested at 2-7 days after treatment with puromycin and inspected for cell viability as described above. Each experiment was performed three times. The number of viable cells was expressed as percentage ± standard error.

Gene Transfer and Selection of Transfectants

Fleshly isolated fetal somatic cells (1 X 107) were electroporated in 500 |Л of HEPES-buffered saline containing 10 |xg of EGFPac using an electroporation system (Gene Pulser II; Bio-Rad Co. Ltd., Hercules, CA) with 750 V/cm, 950 |xF one pulse and 40 msec wavelength. Electroporated cells were then cultured in a 100-mm plastic dish (#3003; Becton Dickinson, Franklin Lakes, NJ) with 10 ml of Dulbecco modified Eagle medium (DMEM) (D5796; Sigma Co. Ltd.) containing 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 in air. After 48 h in culture, 2 ^g/ml of puromycin was added to the medium for selection of cells carrying EGFPac. On the seventh day following selection, surviving cells were grown to confluence and the expression of EGFP was confirmed under a fluorescence microscope as described below. These cells were then transferred into a 60-mm dish (#3004; Becton Dickinson) for nuclear transfer, and the remaining cells were deep frozen using culture medium containing 10% (v/v) DMSO. In total, it took 7 days to obtain the recombinant donor cells. As a control group, freshly isolated fetal somatic cells (1 X 107) were cultured for 2-3 days until confluent and then electroporated in 500 |Л of HEPES-buffered saline containing 10 |xg of EGFPac as described above. The colonies of the recombinant fibroblast cells were transferred into 24-well plates after EGFPac transfections and puromycin selection. These cloned cells were then cultured for 3-4 days until confluent. Finally, these cells were transferred into a 35-mm dish and subcultured for 3-4 days until nuclear transfer. In this group, the total culture period of the recombinant cells was around 16-18 days. Also, freshly isolated fetal somatic cells that had not been subjected to electroporation were also cultured for 2-3 days and then subjected to nuclear transfer or observation for EGFP fluorescence as a positive control for nuclear transfer and as a negative control for EGFPac transfections, respectively.

Nuclear Transfer and Transplantation of Manipulated Embryos to Recipients

Transgenic cloned piglets were produced by nuclear transfer as described previously. Briefly, nuclei from puromycin-selected transfectants or normal fetal somatic cells were each introduced into a single enucleated oocyte by piezo-actuated microinjection. A total of 3212 enucleated oocytes were subjected to nuclear transfer with nuclei from the puromycin-selected transformants. Similarly, a total of 250 enucleated oocytes were transplanted with nuclei from wild-type cells. The reconstructed (nuclear-transplanted) oocytes were then electrically activated and cultivated in PZM3 medium in an atmosphere of 5%CO2, 5%O2, and 90% air at 38.5°C for 2 days until two- to eight-cell stage. The surviving embryos were transferred to the oviducts (30-120 embryos per a surrogate) of an anesthetized surrogate mother (matured LWD; Landrace X Large White X Duroc crosses). A portion of the two- to eight-cell stage embryos were cultivated for 6 days until blastocyst stage and then inspected for EGFP fluorescence, as described below. Embryo transfer was performed nine times using 14 surrogates for the embryos derived from nuclear transplantation of puromycin-selected transfectants. Eleven surrogate mothers were used for embryo transfer of the embryos derived from nuclear transplantation of wild-type cells. These surrogate mothers were previously pseudopregnant by artificial insemination after injection of superovulation-inducing hormones and artificially aborted. After embryo transfer, these mothers were kept under a conventional environment employed for housing pigs and observed daily for confirmation of pregnancy by checking estrus. All of the cloned piglets were delivered by natural birth without any chemical induction.

Observation of EGFP Expression

Expression of EGFP in surviving cells after puromycin selection and nuclear transfer-derived blastocysts was examined under a fluorescence microscope (Olympus, Tokyo, Japan) with DM filters (Olympus). Expression of EGFP in the surface skin of cloned piglets was examined visually using Orange Spectacles (UVP, Upland, CA) under an appropriate excitation light source (wavelength 480 nm) converted from ultraviolet (UV) light (302 nm) using a UV/Blue converter plate (UVP) or in cells isolated from tissues such as the ear under a fluorescence microscope as reported by Okabe et al.. Photographs were taken under the excitation light source with a Sharp Cut Filter (Y-50; Kenko, Tokyo, Japan) using a NIKON D1H digital camera (Tokyo, Japan). No image intensifying procedure was applied to any of the photos.

Transgene Analysis

Genomic DNA was extracted from the primary-cultured somatic cells after gene transfer; tissues (including ear and liver) were obtained either by biopsy from live piglets or from animals that had died shortly after birth, as described by Blin and Stafford.

For PCR analysis, 1 |Л of DNA (0.1 |xg) was dissolved in 24 |Л of PCR buffer (10 mM Tris-HCI, pH 9.0, 50 mM KCI, 1.5 |xM MgC12, and 1% Triton X-100) containing 0.2 mM dNTPs, 0.2 mM primers, and 50 U/ ml Taq polymerase (Applied Biosystems, Foster City, CA). PCR was performed using the following protocol: 96°C for 45 sec, 58°C for 25 sec, and 72°C for 3 min (30 cycles). Primers used were EGFPf2 (5′-gacgtaa-acggccacaagttc-3′) and EGFPr2 (5′-atgccgttcttctgcttgtc-3′) (Fig. 1). This primer set yields a 421-base pair (bp) product; products were analyzed by electrophoresis on a 1.5% agarose gel and visualized under UV illumination after staining gels with ethidium bromide. One nanogram of p-EGFPac vector DNA was used in the PCR reaction as a positive control. For the negative control, 1 |xl of genomic DNA (0.1 |xg) isolated from Landrace liver was also subjected to PCR. ф X 174 phage DNA digested with HincII was used as molecular weight markers.

For genomic Southern blot analysis, genomic DNA (10 |xg) was digested with EcoRI, thereby producing a 0.7-kb fragment containing the EGFP cDNA sequence. After electrophoresis of the enzyme-digested DNA on a 1.0% agarose gel, the DNA was finally transferred onto Hybond-N+ filters (Amersham Pharmacia Biotech, Little Chalfont, England). These filters were hybridized with a 32P-labeled EGFP cDNA probe, then exposed to an Imaging Plate (Fuji Film, Tokyo, Japan) overnight, and finally analyzed with the aid of a FLA-3000G image analyzer (Fuji Film).

Western Blot Analysis

Puromycin-selected transfectants and primary cultured cells derived from cloned transgenic piglets were homogenized in sample buffer (10% sucrose [w/v], 3% SDS [w/v], 60 mM Tris-HCl [pH 6.8)]). After the su-pernatants were treated with pmercaptoethanol, they were separated by electrophoresis under reducing conditions on a 10% polyacrylamide-SDS-gel and transferred to nylon membranes (Immobilon-P; Millipore, Bedford, MA). Blots were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS; 50 mM Tris-HCl [pH 7.4], 150 mM NaCl) and then incubated with anti-GFP antibody (ABR, Golden, CO). After washing with TBS, blots were incubated with horse radish peroxidase (HRP)-linked anti-rabbit IgG in TBS containing nonfat dry milk. Blots were then rewashed with TBS. EGFP proteins were detected by treatment of the membranes with ECL Plus western blotting reagent (Amersham) and subsequent exposure to an x-ray film for several minutes at room temperature.