RESULTS

Puromycin Sensitivity Assay for Fetal Porcine Somatic Cells

To determine the optimal concentration of puromycin for the selection of pac-transfected cells, a puromycin resistance test was performed by culturing fetal porcine somatic cells in a medium containing 0.5-6.0 |xg/ml of puromycin for 7 days. No surviving colonies were obtained when exposed to puromycin at concentrations higher than 2 |xg/ml (Fig. 2A). When cells were grown in medium containing 2 |xg/ml of puromycin, none of the resultant cells survived for more than 6 days (Fig. 2B). We therefore used 2 |xg/ml of puromycin for the selection of cells transfected with EGFPac.

Isolation of Puromycin-Resistant Pig Somatic Cells

Approximately 104 cells survived in the presence of pu-romycin (2 |xg/ml) after gene transfer involving the EGFPac construct. Almost all of these surviving cells exhibited EGFP fluorescence (Fig. 3, A and B). A portion of cells (a total of eight colonies) that had survived after selection with puromycin was then examined for the presence of EGFP cDNA by PCR; the EGFP cDNA was detected in each sample (data not shown). Western blotting analysis using an anti-GFP antibody also confirmed that these surviving cells expressed EGFP protein (which was identified as a 38-kDa band) (data not shown).

Production of Transgenic Somatically Cloned Pigs

To obtain somatic clones in pigs, we transferred the nuclei of EGFP-expressing cells that had been selected with puromycin after gene transfer of EGFPac, to the enucleated oocyte. From a total of 3466 enucleated oocytes receiving nuclei from EGFP-expressing cells, 3212 oocytes survived, and 57.5% of these surviving oocytes developed in vitro to the normal two- to eight-cell stage (Table 1). When a portion of these developing two- to eight-cell stage embryos were cultured up to the blastocyst stage and then inspected for EGFP fluorescence, all embryos tested (5/5) exhibited bright fluorescence over the entire surface of the blastocyst (Fig. 3, C and D). When a total of 1401 enucleated oocytes were implanted with nuclei from untransfected fetal fibroblast cells, 1273 oocytes survived, and 43.0% of these treated oocytes developed in vitro to the normal two- to eight-cell stages (Table 1). However, none (5/5 tested) of the blastocysts developed in vitro from the surviving two- to eight-cell embryos exhibited EGFP fluorescence (data not shown).

From a total of 1680 developed embryos that had been derived from the nuclear transfer of puromycin-resistance cells transferred to 14 surrogate mothers, nine piglets (0.52%) were successfully delivered (Table 1). All of these piglets clearly expressed EGFP throughout their body surface when inspected under UV illumination (data not shown). Unfortunately, eight of the nine piglets died shortly after birth due to unknown reasons, but the remaining male piglet (termed L15-112) survived and appeared healthy. No histological and anatomical abnormalities were noted in any of the dead piglets (data not shown). The L15-112 piglet is now 10 mo old and still appears normal. In the control experiment, four (1.87%) piglets were obtained after transfer of a total of 213 developed embryos, which had previously been subject to nuclear transfer with nuclei of untransfected fetal fibroblast cells, to 11 surrogate mothers (Table 1). Unfortunately, one of the four piglets died accidentally, but the remaining piglets (termed M12-86, M13-11, and M14-42) grew healthily. On the other hand, no piglet was obtained from EGFPac recombinant cells by using the conventional cellular cloning before and after gene transfection (Table 1).

The surviving L15-112 pig (aged 10 mo) clearly expressed the EGFP in a variety of tissues. For example, intense fluorescence was observed in the skin (including hair) (Fig. 4, A and D), and epithelial tissues taken from the snout, oral, nasal mucosa, coronary band, and hoof wall (Fig. 4, E and F) under an excitation light source. To confirm the presence of the EGFPac transgene in somatically cloned pigs, genomic DNA prepared from the ear of live (L15-112) or dead (pig 1, pig 2, and L15-113: from the same litter as L15-112) piglets were subjected to PCR. All the samples tested contained EGFPac (Fig. 5). Southern blotting hybridization analysis also revealed the presence of a hybridization signal in all of the samples tested except from pig 5 (Fig. 6). Genomic DNA was degenerated in pig 5 and could not be obtained from pig 9 because of degradation. The transgene copy number was estimated to be 80 in the L15-112 piglet.
Fih1A Novel Method for the Production-1
FIG. 1. Schematic representation of the EGFPac transgene used to express the puromycin-resistant gene (pac) and enhanced green fluorescent protein (EGFP) cDNA. pEGFPac was constructed by inserting the CAG-EGFP fragment into the pPGK-pac-p(A) cassette vector. Arrows indicate 5′ to 3′ orientation of the cDNA or gene. Two expression units (CAG-EGFP and PGK-pac-p(A)) were aligned in a tail-to-tail manner because, in a preliminary experiment, we noted that gene expression occurred more efficiently in primary-cultured fetal porcine somatic cells transfected by the construct aligned in a tail-to-tail manner than those transfected by the construct aligned in a head-to-tail manner (unpublished). Primers EGFPf2 and EGFPr2, used for the detection of EGFPac introduced into the porcine genome by PCR, are shown below the EGFP cDNA. CMV-IE, Cytomegalovirus immediate-early 1 gene enhancer; CA, chicken pactin promoter; pA, 3′ noncoding region of rabbit pglobin gene including a poly(A) signal; PGK, mouse phosphoglycerate kinase 1 promoter; p(A), mouse phosphoglycerate kinase 1 poly(A) signal.

Fig2A Novel Method for the Production-2
FIG. 2. Puromycin sensitivity assay for fetal porcine somatic cells. A) The effect of puromycin concentration upon survivability of porcine fetal somatic cells. Cells were cultured in medium containing 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, and 6 |xg/ml of puromycin for 7 days. After 7 days of culture, cells were harvested and analyzed for cell viability and luciferase activity. Experiments were performed three times. The number of viable cells (luciferase activity) was expressed as percentage ± standard error. B) The effect of culture period length upon the survivability of porcine fetal somatic cells. Cells were harvested at the indicated time point in medium containing 2 ^g/ml of puromycin, and viability was analyzed with luciferase activity, according to the method described in the Materials and Methods. Experiments were performed three times. The number of viable cells (luciferase activity) was expressed as percentage ± standard error.

Fig3A Novel Method for the Production-3
FIG. 3. EGFP expression in porcine fetal somatic cells (A, B) surviving in the presence of 2 ^g/ml of puromycin for 7 days after transfection with EGFPac and in blastocysts (C, D) derived from embryos transferred with nuclei of the EGFP-expressing cells. Cells and blastocysts were microscopically inspected for EGFP fluorescence under UV illumination. Note that almost all cells in transfected fetal somatic cells and nuclear transplanted blastocysts expressed EGFP strongly. A and C, bright field; B and D, dark field.

Fig4A Novel Method for the Production-5
FIG. 4. The L15-112 piglet derived from enucleated oocytes transferred with nuclei of EGFP-expressing somatic cells. Note that EGFP fluorescence was expressed over the entire body surface of the cloned transgenic piglet. A, C, and D) Bright field; (B, E, and F) under dark field; (G-M) wild-type pig as a negative control.

TABLE 1. Effect of donor nuclei source on transgenic pig production.
tBLE1A Novel Method for the Production-4
a Donor nuclei from transfectants surviving in the presence of puromycin after gene transfer of EGFPac, or from untransfected cells, were used for nuclear transfer experiments. b Percentage of embryos transplanted.
c Percentage of total piglets born. Animals living beyond 1 mo after birth are recorded. d Results of somatic cloning with EGFPac recombinant cells after cellular cloning.

Fig5A Novel Method for the Production-6
FIG. 5. PCR analysis of genomic DNA from the transgenic somatically cloned piglets. Note that all piglets born (L15-112, L15-113, pig 1, and pig 2) possessed the EGFPac transgene in their genome. Lane P, positive control (10 pg of pEGFPac vector); lane N, negative control (100 ng of genomic DNA from Landrace ear); lane pig 1, liver of the pig 1 piglet that died immediately after birth; lane pig 2, liver of the pig 2 piglet that died immediately after birth; lane L15-112, ear of the L15-112 piglet that grew healthy; lane L15-113, liver of the L15-113 piglet that died immediately after birth. M, ф X 174 phage DNA digested with Hincll.

Fig6A Novel Method for the Production-7
FIG. 6. Southern blotting analysis of genomic DNA from somatically cloned transgenic piglets. Note that all piglets born (L15-112, L15-113, pig 1, pig 2, pig 5, pig 6, pig 7, and pig 8) possessed the EGFPac transgene in their genome. Lane N.C., negative control (10 ^g of EcoRI digested wild-type Landrace genomic DNA); lane pig 1 and pig 2, genomic DNA derived from the liver of pig 1 and pig 2 that died immediately after birth; lane L15-112, ear of the L15-112 piglet that grew healthy; lane L15-113, liver of the L15-113 piglet that died immediately after birth; lane pig 58, liver of the pigs 5, 6, 7, and 8 that died immediately after birth.