Production of Nuclear Transfer-Derived Piglets Using Porcine Fetal ...

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BIOLOGY OF REPRODUCTION 69, 1060–1068 (2003) Published online before print 28 May 2003. DOI 10.1095/biolreprod.102.014886

Production of Nuclear Transfer-Derived Piglets Using Porcine Fetal Fibroblasts Transfected with the Enhanced Green Fluorescent Protein1 Sanghwan Hyun,3 Gabsang Lee,3 Daeyoung Kim,3 Hyesoo Kim,3 Sohyun Lee,3 Donghyun Nam,3 Yeonwoo Jeong,3 Sue Kim,3 Soocheong Yeom,3 Sungkeun Kang,3 Jaeyong Han,4 Byeongchun Lee,3 and Woosuk Hwang2,3,4 Department of Theriogenology and Biotechnology,3 College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea School of Agricultural Biotechnology,4 Seoul National University, Suwon 441-744, Korea ABSTRACT A system for somatic cell nuclear transfer (SCNT) was developed and led to the successful production of GFP-transfected piglets. In experiment 1, two groups of SCNT couplets reconstructed with porcine fetal fibroblasts (PFF) and enucleated sow (S) or gilt oocytes (G): 1) received a simultaneous electrical fusion/activation (S-EFA or G-EFA groups), or 2) were electrically fused followed by activation with ionomycin (S-EFIA or G-EFIA groups), or 3) were subjected to electrical fusion and subsequent activation by ionomycin, followed by 6-dimethylaminopurine treatment (S-EFIAD or G-EFIAD groups). The frequency of blastocyst formation was significantly higher in S-EFA (26%) compared with that observed in the other experimental groups (P , 0.05), but not with S-EFIA (23%). Sow oocytes yielded significantly higher cleavage frequencies (68%–69%) and total cell numbers of blastocysts when compared with gilt oocytes, regardless of fusion/activation methods (P , 0.05). However, the ratio of inner cell mass (ICM)/total cells in G-EFA and S-EFA was significantly lower than in the other groups (P , 0.05). In experiment 2, SCNT couplets reconstructed with PFF cultured in the presence or absence of serum and enucleated sow oocytes were subjected to EFA. There were no effects of serum starvation on cell-cycle synchronization, developmental competence, total cell numbers, and ratio of ICM/total cells. In experiment 3, SCNT couplets reconstructed with PFF transfected with an enhanced green fluorescence protein (EGFP) gene using FuGENE6 and enucleated sow oocytes were subjected to EFA and cultured for 7 days. Expression frequencies of GFP gene during development were 100%, 78%, 72%, 71%, and 70% in fused, two-cell, four to eight cells, morulae, and blastocysts, respectively. In experiment 4, SCNT embryos derived from different recipient cytoplasts (sows or gilts) and donor karyoplasts (PFF or GFP-transfected) were subjected to EFA and transferred to the oviducts of surrogates. The pregnancy rates in SCNT embryos derived from sow oocytes (66%–69%) were higher than those with gilt oocytes (23%–27%) regardless of donor cell types. One live offspring from GFP-SCNT embryos and two from PFFSCNT embryos were delivered. Microsatellite analysis confirmed Supported by grants from the Advanced Backbone IT Technology Development (IMT2000-C1-1) and the Korean Ministry of Science and Technology (1998-019-G00021). The authors acknowledge a graduate fellowship provided by the Ministry of Education through BK21 program. 2 Correspondence: Woosuk Hwang, Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151–742, Korea. FAX: 822 884 1902; e-mail: [email protected] 1

Received: 19 December 2002. First decision: 20 January 2003. Accepted: 8 May 2003. Q 2003 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

that the clones were genetically identical to the donor cells and polymerase chain reaction (PCR) from genomic DNA of cloned piglets and subsequent southern blot analysis confirmed the integration of EGFP gene into chromosomes.

early development, embryo, gamete biology, ovary

INTRODUCTION

Production of cloned and transgenic pigs by somatic cell nuclear transfer (SCNT) has unlimited value for developing critical biotechnology such as xenotransplantation [1, 2]. Various efforts have been made to establish this technology [3], and live piglets have been delivered after transfer of SCNT embryos [4–6]. However, the viability of porcine SCNT embryos is poor, with an extremely low rate of cloned piglet production. Studies have demonstrated that many factors are involved in the development of porcine SCNT embryos [7]. The factors include donor cell types, recipient oocytes, fusion/activation methods, and in vitro culture system. For example, oocytes collected from superovulated prepubertal gilts exhibit a decreased rate of fertilization and viability in vivo compared with young postpubertal sow oocytes [8, 9], suggesting the importance of source of oocytes for the developmental competence of embryos. Various fusion/activation protocols have been reported for the production of porcine SCNT embryos, including delayed chemical activation after electrical fusion [4] and simultaneous electrical fusion/activation treatment [10–12]. The possibility of transfecting the cells used as donor karyoplasts provides a powerful tool for the production of transgenic livestock, which should, after further improvement of the technique, result in higher production efficiency compared with traditional pronuclear injection [12–16]. Since first introduced as an expression marker by Chalfie and colleagues in 1994 [17], a fusion protein (green fluorescent protein, GFP) or a peptide tagged with GFP is being used as a marker to follow in vivo gene expression and real-time protein localization [18, 19]. In several species, including pigs, enhanced green fluorescent protein (EGFP) gene was successfully used as an indicator without any adverse biological effects on in vitro development of transfected embryos [11, 20–22]. In addition, transgenic offspring carrying the GFP gene have been successfully produced in mice, monkeys, and pigs [16, 20, 22]. The success in selecting and producing transgenic offspring using GFP as a marker has paved the way for GFP use in transgenic experimentation. With the aid of this virtually ideal transgenic marker, progress in developing efficient gene delivery systems will be greatly accelerated.

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Considering the virtually unlimited value of transgenic pigs in critical biotechnology applications, this study was conducted to establish a system for the production of transgenic pigs using SCNT of GFP-transfected cells into enucleated oocytes.

of incubation at room temperature, 101 ml DNA-medium mixtures were added into 2 ml of cell culture medium. The cells were cultured for 2–3 days until confluency and passaged once to achieve stable integration of the gene into chromosomes before use for SCNT.

MATERIALS AND METHODS

At 42 h IVM, a cumulus-free oocyte was held with a holding micropipette (110-mm inner diameter) and the zona pellucida was partially dissected with a fine glass needle to make a slit near the first polar body. The first polar body and adjacent cytoplasm presumably containing the metaphase-II chromosomes were extruded by squeezing with the needle. Oocytes were enucleated in Hepes-buffered NCSU-23 supplemented with 0.3% BSA and 7.5 mg/ml cytochalasin B (Sigma-Aldrich Corp.). After enucleation, oocytes were stained with 5 mg/ml bisbenzimide (Hoechst 33342, Sigma-Aldrich Corp.) for 5 min and observed under an inverted microscope equipped with epifluorescence. Oocytes still containing DNA materials were excluded from experiments. Trypsinized nontransfected or EGFP-transfected single cells with a smooth surface were selected under an inverted microscope equipped with a GFP filter (wavelength: exciting 489 nm and emission 508 nm) and were transferred into the perivitelline space of enucleated sow or gilt oocytes. These couplets were equilibrated with 0.3 M mannitol solution containing 0.5 mM Hepes, 0.1 mM CaCl2, and 0.1 mM MgCl2 for 4 min and transferred to a chamber containing two electrodes that were overlaid with fusion and activation solution. Couplets were fused with a single DC pulse of 2.0 kV/cm for 30 msec using a BTX Electro-Cell Manipulator 2001 (BTX, Inc., San Diego, CA) and activated as described in experiment 1. Activated oocytes were washed three times with NCSU-23 [24] supplemented with 4 mg/ml fatty acidfree BSA (Sigma-Aldrich Corp.), and placed in 25 ml microdrops (5–7 oocytes per drop) of NCSU-23 under mineral oil and cultured at 398C, 5% CO2, 7% O2, and 88% N2. The reconstructed embryos were cultured for 7 days after activation.

Collection of Oocytes and In Vitro Maturation Because the local slaughterhouse has one working line for sows and another for gilts, ovaries from prepubertal gilts and sows were collected by two investigators simultaneously and transported to the laboratory in physiological saline at 30–358C within 2 h of slaughter. Follicular fluid and cumulus-oocytes complexes (COCs) from follicles 5–6 mm in diameter were aspirated using an 18-gauge needle attached to a 5-ml disposable syringe. Compact COCs were selected and washed six times in Hepesbuffered TCM-199. The basic medium used was TCM-199 (Life Technologies, Rockville, MD). The oocyte maturation medium was modified TCM-199 [23] supplemented with 10 ng/ml epidermal growth factor (Sigma-Aldrich Corp., St Louis, MO), 10 IU/ml eCG (Sigma-Aldrich Corp.), 10 IU/ml hCG (Sigma-Aldrich Corp.), and 10% (v/v) porcine follicular fluid. Porcine follicular fluid was aspirated from 6–8-mm diameter antral follicles from prepubertal gilt ovaries. After centrifuging at 1600 3 g for 30 min, supernatants were collected, filtered sequentially through 1.2-mm and 0.45-mm syringe filters (Gelman Sciences, Ann Arbor, MI) and stored at 2308C until use. A group of 50 COCs was cultured in 500-ml of each in vitro maturation (IVM) medium at 398C in a humidified atmosphere of 5% CO2 and 95% air. After culturing for 22 h, COCs were transferred to eCG- and hCG-free IVM medium and cultured for another 20 h. At the end of the maturation culture, oocytes were freed from cumulus cells by repeated pipetting in the same IVM medium containing 0.5 mg/ml hyaluronidase (Sigma-Aldrich Corp.) for 1 min.

Preparation of Fetal Fibroblast Cell Fibroblasts were isolated from pig fetuses on Day 30 of gestation, as previously described [6]. Briefly, collected fetuses were washed three times with Ca21- and Mg21-free PBS (DPBS-, Life Technologies). The heads and internal organs were removed using iris scissors and forceps. The remnants were washed twice in DPBS-, minced with a surgical blade on a 100-mm culture dish, and followed by dissociation with 0.25% (v/v) trypsin-EDTA (Life Technologies) containing Dulbecco modified Eagle medium (DMEM, Life Technologies) at 398C for 1–2 h. After centrifuging three times at 300 3 g for 10 min, cell pellets were subsequently seeded onto 100-mm plastic culture dishes (Becton Dickinson, Lincoln Park, NJ) and cultured for 6–8 days in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS; Life Technologies), 1 mM sodium pyruvate (SigmaAldrich Corp.), 1% (v/v) nonessential amino acids (Life Technologies), and 10 mg/ml penicillin-streptomycin (Sigma-Aldrich Corp.) in a humidified atmosphere of 5% CO2 and 95% air. After removal of unattached clumps of cells or explants, attached cells were further cultured until confluent, subcultured at intervals of 5–7 days by trypsinization for 5 min using 0.1% trypsin and 0.02% EDTA and stored after two passages in freezing medium in liquid nitrogen at 21928C. The freezing medium consisted of 80% (v/v) DMEM, 10% (v/v) dimethyl sulfoxide (Sigma-Aldrich Corp.) and 10% (v/v) FBS. After thawing, cells were cultured in DMEM supplemented with 10% FBS to approximately 70% confluency (passage 3–5). For serum starvation, cells were cultured for 3 days in serum-starved DMEM supplemented with 0.2% FBS prior to SCNT. Donor cells to be subjected for flow cytometry sorting were trypsinized, centrifuged, and resuspended in 1 ml of PBS. After resuspension, cells were incubated with DNase-free RNase A (Life Technologies) for 30 min at 378C, followed by staining with 1 mg/ml propidium iodide for 10 min at 258C before flow cytometry analysis.

Somatic Cell Nuclear Transfer and Culture

Evaluation of Blastocyst Quality The quality of blastocysts was assessed by differential staining of the inner cell mass (ICM) and the trophectoderm (TE) according to the modified staining procedure of Thouas et al. [25]. Briefly, trophectoderm cells of blastocysts at 7 days were stained with the fluorochrome propidium iodide (Sigma-Aldrich Co.) after treatment with permeabilizing solution containing the ionic detergent Triton X-100 (Sigma-Aldrich Corp.). Blastocysts were then incubated in a second solution containing 100% ethanol (for fixation) and bisbenzimide (Sigma-Aldrich Corp.). Fixed and stained whole blastocysts were mounted and assessed for cell number using ultraviolet fluorescent microscopy.

Microsatellite Analysis Parentage analysis was performed on the piglets produced by SCNT and the surrogate recipient females to confirm genetic identity with the donor cells used for SCNT. Tissue fragments were obtained from an ear punch or tail clipping of each newborn piglet and from recipient females. The tissue fragments and trypsinized donor cells were incubated with a lysis buffer (0.05 M Tris [pH 8.0], 0.05 M EDTA [pH 8.0], 0.5% SDS) supplemented with 400 g proteinase K overnight, followed by phenol extraction and ethanol precipitation. The isolated genomic DNA samples were dissolved in 50 ml TE and used for microsatellite assay using eight porcine DNA microsatellite markers (S0086, S0230, SW902, S0007, S0313, SW61, S0005, and S0164) labeled with one of the fluorescent dyes FAM, TET, or HEX [26]. Length variations were assayed by polymerase chain reaction (PCR) amplification with fluorescently labeled locus-specific primers and PAGE on an automated DNA sequencer (ABI 373; Applied Biosystems, Foster City, CA). Proprietary software (GeneScan and Genotyper; Applied Biosystems) was used to estimate PCR product size in nucleotide.

Transfection of EGFP Gene into Fetal Fibroblasts

Detection of EGFP Gene in Cloned Piglets

The plasmid pEGFP-N1 encoding a red-shifted variant of wild-type GFP that has been optimized for brighter fluorescence and higher expression in mammalian cells was purchased from Clontech Laboratories, Inc. (Palo Alto, CA). The day before transfection, confluent fetal fibroblasts (at passage 3–5) were trypsinized, counted, and plated into 35-mm culture dishes to reach 80% confluency on the day of transfection. One microliter (1 mg) of pEGFP-N1 and 3 ml of FuGENE-6 (Roche Diagnostics, Indianapolis, IN) were diluted with 97 ml of serum-free DMEM. After 15 min

In order to detect the EGFP gene in cloned piglets, genomic DNA was extracted as described above and PCR amplification was performed using one set of primers for EGFP. One primer set was designed based on the published sequence of GFP that amplifies a fragment of 431 base pairs (bp). Primers for GFP were sense, 59-GCG ATG CCA CCT ACG GCA AGC TGA-3, and antisense, 39-GAG CTG CAC GCT GCC GTC CTC GAT-59. The genomic DNA (300 ng) was amplified in a 50-ml PCR reaction containing 2.5 units Hotstart Taq polymerase (Qiagen, Hilden, Ge-

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TABLE 1. Preimplantation development of porcine cloned embryos and allocations of cells (mean 6 SEM) to inner cell mass (ICM) and total cell in porcine blastocysts produced by different reconstruction methods using enucleated sow or gilt oocytes and fetal fibroblasts in experiment 1. No. and (%) of cloned embryos developed to No. and (%) of oocytes Source (no.) of Fusion/ enucleated activation oocytes methods Gilts (932) Sows (503)

EFAa EFIAb EFIADc EFAa EFIAb EFIADc

Stage shown

Used for Successfully NT fused

Two-cell [48 h]

250 250 250 137 142 130

144 143 147 94 98 89

186 183 184 104 105 99

(74) (73) (74) (76) (74) (76)

(58)d (57)d (59)d (69)e (69)e (68)e

Eight-cell [96-h] 107 104 61 69 66 56

(43)df (42)df (24)e (50)d (46)df (38)f

Cell no. of blastocysts

Morula [144 h] 76 72 45 52 51 39

(30)df (29)df (18)e (38)f (36)df (27)de

Blastocyst [168 h] 45 42 29 36 33 28

(18)df (17)df (12)f (26)e (23)de (16)df

ICM (no. blastocysts examined) 14.2 17.3 17.4 18.7 21.1 21.6

6 6 6 6 6 6

3.6 5.7 3.8 5.9 8.1 7.3

(45)d (42)e (29)e (36)ef (33)f (21)f

Total 48.4 46.8 48.5 61.1 59.5 60.0

6 6 6 6 6 6

16.6d 12.7d 15.3d 16.9e 20.2e 17.2e

ICM/ total cells 31.7 36.9 37.9 31.6 36.2 36.8

6 6 6 6 6 6

9.4d 6.6e 9.0e 10.5d 8.1e 8.7c

EFA, Electrical fusion and activation simultaneously (single DC pulse of 2.0 kV/cm for 30 msec). EFIA, Electrical fusion followed by ionomycin activation 2 h later. c EFIAD, Electrical fusion followed by ionomycin activation 2 h later and then incubation with 6-DMAP for 2 h. def Within the same column, values with different superscripts differed significantly (P , 0.05). a

b

many) and its buffer, 1.5 mM MgCl2, 2 mM deoxy-NTP, and 50 pmol specific primers. PCR amplification was carried out for 35 cycles with denaturing for 1 min at 948C, annealing for 35 sec at 588C, extension for 90 sec at 728C, and a final extension for 15 min at 728C. Amplified PCR products were subjected to Southern blot analysis. Ten microliters of PCR products were fractionated on a 1.5% agarose gel and stained with ethidium bromide. The PCR products were transferred to a nylon membrane and hybridized with digoxigenin-labeled 621-bp cDNA probe for EGFP (accession number: U55762, 719–1339) following the manufacturer’s recommended procedure (Roche Molecular Biochemicals, Mannheim, Germany). After washing, the membranes were exposed to Hyperfilm ECL (Amersham-Pharmacia Biotech., Little Chalfont, UK). The PCR products isolated from gel were cloned into pCRII vector using the TA Topo Cloning Kit (Life Technologies) and were sequenced using an automated DNA sequencer (ABI 373, Applied Biosystems).

Surgical Embryo Transfer and Pregnancy Diagnosis Potential surrogate gilts at .8 mo of age were checked for estrus twice a day. All SCNT embryos were transferred to the oviduct of the naturally cycling gilts on the first day of standing estrus. Surrogates were injected with thiopental sodium (Daihan Pharm. Co., Seoul, South Korea) intravenously and anesthesia was maintained with Isoflurane (Hana Pharm. Co., Kyonggi-Do, South Korea). The SCNT embryos (150 embryos/surrogate) were loaded into a GIFT catheter (KGIFT-1010; Spencer, IN) and deposited into the oviduct of the surrogate after midventral laparotomy. Examination of the ovaries during embryo transfer confirmed that none of the surrogates had completed ovulation. Nonreturn surrogates were checked for pregnancy by transabdominal ultrasound examination at Day 25 after embryo transfer and at 2 weeks.

Experimental Design In experiment 1, the effect of donor oocytes (sow or gilt oocytes) and methods for fusion/activation on developmental competence of SCNT embryos and number of cell per blastocyst were evaluated. The SCNT couplets derived from porcine fetal fibroblasts (PFF) were subjected to different fusion/activation protocols; 1) two groups of couplets derived from either enucleated gilt (G) or sow (S) oocytes received a simultaneous electrical fusion/activation (G-EFA and S-EFA groups, respectively), or 2) were electrically fused and cultured for 2 h in NCSU-23, followed by activation with 15 mM calcium ionomycin for 4 min (G-EFIA and S-EFIA groups, respectively), or 3) were subjected to electrical fusion and subsequent activation by 15 mM calcium ionomycin for 4 min, followed by 6-dimethylaminopurine (6-DMAP) treatment (G-EFIAD and S-EFIAD groups, respectively) for 2 h. In experiment 2, the effect of serum starvation on cell-cycle synchronization, SCNT embryo development, and the cell number in blastocysts were monitored. The PFF were cultured in serum-fed DMEM with 10% FBS or serum-starved DMEM with 0.2% FBS for 3 days and used for SCNT. As a result of experiment 1, sow oocytes and EFA were subsequently used as optimal recipient cytoplasts and EFA as the fusion/activation method. In experiment 3, GFP-transfected PFF cultured in serum-fed media were transferred to enucleated

sow oocytes and subjected to EFA as described in experiment 1. The expression rate of GFP during embryo development was monitored. In experiment 4, in vivo viability of SCNT embryos was examined after embryo transfer. The SCNT embryos were reconstructed with different sources of donor karyoplasts (nontransfected PFF or GFP-transfected PFF) cultured in serum-fed media and recipient cytoplasts (gilt or sow oocytes), fused and activated simultaneously (EFA) as described in experiment 1. The SCNT embryos (150 embryos/surrogate) were transferred to one oviduct of gilts .8 mo of age within 8 h after fusion/activation. The rate of pregnancy and delivery and the clinical and pathological findings on delivered piglets were examined.

Statistical Analysis Data were analyzed using the statistical analysis system (SAS) program. Random distribution of SCNT embryos was made in each experimental group and experiments were replicated at least six times. Parametric analysis of the means between two or more populations was analyzed by an ANOVA followed by multiple pairwise comparisons using a Duncan test. Experiments 1 and 4 were first analyzed for interaction among experimental parameters. As no interaction was found, the data were further analyzed by an ANOVA followed by multiple pairwise comparisons using a Duncan test. Differences of P , 0.05 were considered significant.

RESULTS

In experiment 1, the use of sow oocytes yielded better results compared with gilt oocytes in embryo development to 2 cells (68% to 69% vs. 57% to 59%, respectively) regardless of fusion/activation methods (Table 1). When compared between fusion/activation methods and oocyte sources, the frequency of blastocyst formation was significantly higher in S-EFA (26%) compared with that observed in the other experimental groups (P , 0.05) but not to S-EFIA (23%). Embryo developmental competence was not different between the EFA and EFIA groups in all experimental parameters monitored. As shown in Table 1, total cell numbers of blastocysts derived from sow oocytes (S-EFA, SEFIA, and S-EFIAD: 61.1 6 16.9, 59.5 6 20.2, and 60.0 6 17.2, respectively) were significantly higher than those of blastocysts from gilt oocytes (G-EFA, G-EFIA, and GEFIAD: 48.4 6 16.6, 46.8 6 12.7 and 48.5 6 15.3, respectively) (P , 0.05). Regardless of fusion/activation methods, there was no significant difference in total cell numbers among blastocysts derived from gilt (G-EFA, GEFIA, and G-EFIAD) or sow oocytes (S-EFA, S-EFIA, and S-EFIAD). However, ratios of ICM/total cell in the G-EFA

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TABLE 2. Effects of serum starvation culture of porcine fetal fibroblasts on preimplantation development of porcine cloned embryos reconstructed with enucleated sow oocytes and allocation of cells (mean 6 SEM) to inner cell mass (ICM) and total cell in porcine blastocysts obtained from experiment 2. No. and (%) of cloned embryosa developed to No. and (%) of oocytes Type of cells Starvation culture PFFb a b

Serum-starved Serum-fed

Stage shown

Cell no. of blastocysts

Used for Successfully NT fused

Two-cell [48 h]

Eight-cell [96-h]

Morula [144 h]

Blastocyst [168 h]

ICM (no. blastocysts examined)

Total

ICM/ total cells

144 133

95 (66) 92 (69)

65 (45) 67 (50)

54 (38) 47 (35)

31 (22) 32 (24)

18.6 6 5.4 (31) 18.4 6 6.1 (32)

60.2 6 17.4 61.0 6 16.3

32.2 6 10.3 30.8 6 10.3

102 (71) 101 (76)

Fusion and activation of cloned embryos were performed simultaneously by electrical stimulation (single DC pulse of 2.0 kV/cm for 30 msec). PFF, Porcine fetal fibroblasts.

(31.7 6 9.4) and S-EFA groups (31.6 6 10.5) were significantly lower than in the other treatment groups (P , 0.05). In experiment 2, no significant differences in the stage of cell cycle in serum-fed or -starved porcine fetal fibroblasts (G0/G1 phase, 75% to 76%; S phase, 8% to 9%; and G2/M phase, 14% to 16%) were observed. Serum starvation did not affect fusion rate, embryo development rate from two-cell to blastocyst stage, total cell numbers, and ratio of ICM/total cells in blastocysts (Table 2). In experiment 3, after SCNT, GFP expression rates were 100%, 78.2%, 71.4%, 71.1%, and 70.0% in fused, two, four to eight cell, morula, and blastocyst stages, respectively (Fig. 1 and Table 3). In experiment 4, SCNT embryos reconstructed with sow oocytes gave better pregnancy rates (67% to 69%) than gilt oocytes (23% to 27%) when diagnosed at Days 25 and 39 after embryo transfer, regardless of GFP gene transfection into donor cells (P , 0.05). The GFP transfection had no effect on the pregnancy rate of SCNT embryos reconstructed with sow and gilt oocytes compared with that of SCNT embryos reconstructed with nontransfected PFF. A significantly higher pregnancy maintenance rate (P , 0.05) was observed in SCNT embryos reconstructed with sow oocytes and GFP transfected cell (54%) compared with that of gilt oocytes (0% and 18%), but not with SCNT embryos derived from sow oocytes and PFF cells (40%). At term, only SCNT embryos reconstructed with sow oocytes delivered cloned (Fig. 2A) and/or transgenic piglets (Fig. 2B). Two live births and one stillbirth from PFF and one live birth and one stillbirth from GFP-transfected PFF were produced from each surrogate. Microsatellite analysis confirmed that the clones were genetically identical to the donor PFF (Table 4). Analysis of genomic DNA by PCR-Southern blot assay revealed the integration of pEGFP gene in live and dead transgenic cloned piglets (Fig. 3). The possibility of cross-contamination was ruled out because no PCR products were observed and detected in the negative control [distilled water (DW) or without template, Tm (2)] by ethidium bromide staining and Southern blot analysis. The PCR products were gel purified, cloned, and sequenced. Sequence analysis confirmed the presence of EGFP gene from live or dead GFP transgenic cloned piglets (data not shown). DISCUSSION

Transgenic farm animals are of great value for research and commercial purposes, and animal donors such as pigs could provide an alternative source of organs for transplantation. In this study, to establish an efficient production system for transgenic cloned pigs, different sources of oocytes

and methods for fusion/activation were tested, and it was found that simultaneous fusion/activation with sow oocytes yield the best developmental competence of SCNT embryos. In addition, it was demonstrated that serum starvation of donor cells had no effect on cell-cycle synchronization, the development and cell number of SCNT embryos. Finally, after embryo transfer, healthy cloned and transgenic cloned piglets were born and their genetic origin was confirmed by microsatellite and genomic PCR-Southern blot assay. In pigs, it was suggested that the source of oocytes is important for the developmental competence of embryos [8, 9]. Previously, we reported the influence of the source of oocytes on the development ability of SCNT embryos, showing that sow oocytes have a greater developmental competence than gilt oocytes [27]. As shown in Table 1, SCNT embryos derived from sow oocytes yielded higher development ability and total cell number than those from gilt oocytes. This result was also confirmed by in vivo viability after surgical embryo transfer, showing that SCNT embryos derived from sow oocytes were significantly more viable than embryos from gilt oocytes (Table 5). In agreement with our results, Marchal et al. [28] demonstrated that adult oocytes were more meiotically competent than their prepubertal gilt counterparts. The present result is also supported by the findings that more immature and more aneuploid oocytes are ovulated by gilts [29] and that gilt oocytes took longer than sow oocytes to mature to second metaphase of meiosis [30]. Betthauser et al. [4] reported that delayed activation with ionomycin 1 6-DMAP is better than electrical activation protocols for producing porcine SCNT embryos. 6-DMAP is commonly used during the postactivation phase for producing SCNT embryos in vitro in several species, including pigs. However, the present study demonstrated that a simultaneous electrical fusion/activation protocol is simple and efficient for producing porcine SCNT embryos, indicating that no further chemical stimulation such as ionomycin and 6-DMAP is necessary for postactivation of porcine SCNT embryos (Table 1). In agreement with our results, Lai et al. reported the successful production of a-1,3galactosyltransferase knockout piglets by SCNT using electrical stimulation alone to activate porcine SCNT embryos [12]. In this study, the ratio of ICM to total cells in EFA was lower than that of EFIA and EFIAD, regardless of oocyte sources (Table 1). This ratio in blastocysts derived from EFA-treated SCNT embryos was similar to that of IVF-blastocysts (data not shown). This result indicates that the delayed activation protocol (EFIA and EFIAD in sow or gilt oocytes) may induce disproportionate cell al-

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FIG. 1. SCNT embryo development from fusion to blastocyst stages. Fusion time was considered to be 0 h. A) Injection of EGFP transfected cells into enucleated oocytes (3100). B) Reconstructed oocyte before fusion (3100). C) Fused embryo at 1 h (3400). D) Two-cell stage at 26 h (3100). E) Fourcell stage at 50 h (3100). F) Eight-cell stage at 74 h (3100). G) Blastocyst stage at 146 h (3200). Bars 5 100 mm.

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No. expressing pEGFP/total no. (%) at each stage shown Fused

Two-cell

Four- to eight-cell

Morula

Blastocysts

127/127 (100)

90/115 (78)

60/84 (72)

42/59 (71)

28/40 (70)

locations in SCNT embryos, leading to reduced subsequent developmental potential. The cell-cycle stage is a subject of debate in the production of cloned animals by SCNT because no system thus far provides 100% synchronization of cells in a certain stage [31] and therefore the use of cells in the same cellcycle stages in SCNT procedures cannot be guaranteed. Whereas some researchers assert that the use of cells in G0

is required for complete reprogramming [32–34], others used cycling donor cells in presumptive G1 and obtained offspring [14]. In this study, we investigated whether serum starvation, which is a commonly used method to synchronize cell cycle, could improve the SCNT embryo development. Serum starvation did not affect cell-cycle synchronization of donor cells, developmental competence of SCNT embryos, and the number of ICM, TE, and total cells FIG. 2. Production of nuclear transfer-derived piglets by somatic cell nuclear transfer of porcine fetal fibroblasts transfected with an enhanced green fluorescent protein (EGFP) into enucleated gilt or sow oocytes. A total of 150 reconstructed embryos were surgically transferred to the oviduct of each surrogate over 8 mo of age. A) GFP-cloned piglet. B) PFF-cloned piglets.

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TABLE 4. In vivo viability of porcine transgenic cloned embryos according to oocyte source and types of donor cell after embryo transfer in experiment 4.

Oocyte source Gilts Sows

Donor cell type PFF GFP PFF GFP

No. of surrogates 13 11 15 13

Pregnancy status (%) of surrogates

No. (%) of pregnant surrogates

Abortion

(23.0)a

3 3 (27.2)a 10 (66.6)b 9 (69.2)b

3 1 3 1

Ongoing

Parturition

(0)a

(23.1) (9.1) (20.0) (7.7)

0 2 (18.0)ab 6 (40.0)bc 7 (53.8)c

0 0 1 1

(0) (0) (6.7)d (7.7)e

Within the same column, values with different superscripts differed significantly (P , 0.05). Two live births and one stillbirth from PFF were produced. e One live birth and one stillbirth from GFP-transfected PFF were produced. a,b,c d

in SCNT blastocysts (Table 2). Taken together, our results suggest that cell-cycle synchronization of donor cells by serum starvation is not required for the production of porcine SCNT embryos. The objective of gene transfer is to produce animals having a stable incorporation of foreign DNA in the germ line that will serve as founder stock to produce many offspring carrying a desirable gene(s). A desired gene can be transferred into the somatic cell by infection with a viral vector or by electroporation with lipid (liposome) or nonlipid (polymer) reagents carrying DNA. For instance, Park et al. [16] used a retroviral vector to produce GFP cloned piglets and Lai et al. [12] applied electroporation to produce a1,3-galactosyltransferase knockout cloned piglets. In this study, we introduced a GFP gene by liposome-mediated gene transfer to produce transgenic cloned piglets by SCNT because liposomes and polymers have been thoroughly used in transfection experiments for cultured cells and in vivo for organs (lung, liver, tumor cells) [35]. Our preliminary studies showed that FuGENE-6R was the most efficient lipid carrier among liposome-mediated transfection reagents tested. As shown in Table 4, GFP-transfected cells were used for SCNT and GFP expression rates were evaluated according to embryo development. Although not all SCNT embryos expressed the GFP gene, it was expressed in all stages of embryo development as seen under UV light. After fusion, GFP was expressed in all fused embryos, but the expression rates were decreased to 78.2% at the two-cell stage, but this stabilized to 70% to 71% between the four- to eight-cell and blastocyst stages. The PCR amplification of GFP gene using genomic DNA of a GFPexpressing blastocyst confirmed the chromosomal integration of GFP gene in blastocysts (data not shown). Using gilt oocytes matured in TCM-199, Park et al. [16] reported 9.3% GFP expression rate in blastocysts/total embryos reconstructed with cumulus cells that were GFP transfected

using a retroviral vector. Although that result cannot be compared directly due to different culture systems and SCNT procedures, the present study showed higher rates of GFP expression rate in blastocysts/total embryos (28/168, 16.6%) derived from couplets reconstructed with the liposome-mediated transfected PFF. The higher GFP-SCNT embryo development achieved in our study may be due to a number of factors. First, the use of sow oocytes could improve the developmental competence of GFP-SCNT embryos. It has been reported that, for SCNT, ovaries are mainly obtained from gilts because of limited availability of sow ovaries and economics [36]. For this reason, Ku¨hholzer et al. [36] used for their experiments commercially available sow oocytes shipped overnight in TCM-Hepes medium. In contrast, in this study, fresh sow oocytes were cultured in Hepes-free IVM medium at 398C, 5% CO2 in a humidified air atmosphere because sow ovaries were freely available every day. Second, GFP-expressing cells were selected without antibiotics and used for SCNT. For the production of transgenic cells, various selection markers have been employed, including antibiotics and GFP with different actions and advantages [17, 37]. Although antibiotics have been used successfully in generating transgenic cells, they induce cellular damage, senescence, and chromosomal abnormality after long-term selection of somatic cells [37]. Third, separate processing of enucleation, injection of donor cells, and fusion/activation by three investigators shortened the time of exposure of embryos to ambient atmospheric (air) conditions and to the SCNT procedure compared with SCNT procedures done by a single investigator. Taken together, our results suggest that the GFP transfection system without establishing stable somatic cell lines can be used for the production of transgenic SCNT embryos and cloned piglets. To deliver cloned piglets, age and estrous stage of surTABLE 5. Microsatellite (MS) analysis of donor cell lines, surrogate, and offspring.a Surrogate GFP-2 No. of GFP donor cell mother GFP-1 (alive) (stillbirth) MS primer Peak 1 Peak 2 Peak 1 Peak 2 Peak 1 Peak 2 Peak 1 Peak 2

FIG. 3. PCR and Southern blot of the EGFP insert in cloned piglets. Genomic DNA was subjected to polymerase chain reaction (PCR) amplification using a specific primer set for enhanced green fluorescent protein (EGFP). The expected products were observed on an ethidium bromide-stained gel (top panel). The PCR products were transferred onto a nylon membrane and hybridized with a digoxigenin-labeled 621-base pair (bp) EGFP cDNA probe (bottom panel). DW, Distilled water (negative control). A) Normal porcine fetal fibroblasts. B) GFP-transfected fetal fibroblasts. C) Surrogate. D) Cloned piglet 2. E) Cloned piglet 1. F) pEGFPN1.

S0086 S0230 SW902 S0007 S0313 SW61 S0005 S0164

219 300 199 188 161 254 236 226

169

195 296 200 173 165 245 228 245

219 304 180 169 254 232

219 300 199 188 161 254 236 226

169

219 300 199 188 161 254 236 226

169

a For each microsatellite marker, genotype was determined by size (base pairs). Litter 2 results from S0086, S0230, SW902, S0007, S0313, SW61, S0005, and S0164 provide the strongest support for the genetic identity of the donor cells and the nuclear transfer piglets.

PRODUCTION OF TRANSGENIC CLONED PIGLETS

rogates should be considered. In a preliminary study, we performed surgical ET into various age and ovulation stage of surrogates and found that ET to surrogates at the age of more than 8 mo and an earlier stage of the estrous cycle than the embryos themselves are needed for pregnancy (data not shown). With this approach, high pregnancy rates (66% to 69%) were obtained in this study after transfer of SCNT embryo reconstructed with enucleated sow oocytes. In agreement with our results, Polge demonstrated that asynchronous ET was more efficient for pregnancy in pigs [38]. The present result is also supported by the findings that noncompleted ovulation of surrogates is the optimal stage for embryo transfer of in vitro produced one-cell stage in manipulated [38], pronuclear microinjected [39], and SCNT-derived embryos [12]. In the present study, at term, two live births and one stillbirth from PFF and one live birth and one stillbirth from GFP transfected PFF were produced. These piglets had normal birth weights (PFF-1 [1.2 kg], PFF-2 [1.4 kg], and GFP-1 [1.4 kg]), which are similar to those from our sow herds (1.3 kg). Initial physical examination of healthy piglets revealed no abnormalities. Also, dead piglets revealed no abnormalities throughout postmortem analysis. In conclusion, the present study shows that 1) sow oocytes are better recipient cytoplasts for SCNT than gilt oocytes, 2) electrical stimulation alone is sufficient for the activation of SCNT embryos, 3) cells transfected with GFP using FuGENE-6 can be used for the production of transgenic piglets, and 4) cell-cycle synchronization of donor cells by serum starvation is not required for the production of normal or GFP transgenic cloned piglets. ACKNOWLEDGMENT We thank Dr. Barry D. Bavister for his valuable editing of the manuscript.

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