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Reproductive Technology |
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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, embryo, gamete biology, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 30–35°C 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 Hepes-buffered 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 x g for 30 min, supernatants were collected, filtered sequentially through 1.2-µm and 0.45-µm syringe filters (Gelman Sciences, Ann Arbor, MI) and stored at -30°C until use. A group of 50 COCs was cultured in 500-µl of each in vitro maturation (IVM) medium at 39°C 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 Ca2+- and Mg2+-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 39°C for 1–2 h. After centrifuging three times at 300 x 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 (Sigma-Aldrich Corp.), 1% (v/v) nonessential amino acids (Life Technologies), and 10 µg/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 -192°C. 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 37°C, followed by staining with 1 mg/ml propidium iodide for 10 min at 25°C before flow cytometry analysis.
Transfection of EGFP Gene into Fetal Fibroblasts
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 µg) of pEGFP-N1 and 3 µl of FuGENE-6 (Roche Diagnostics, Indianapolis, IN) were diluted with 97 µl of serum-free DMEM. After 15 min of incubation at room temperature, 101 µl 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.
Somatic Cell Nuclear Transfer and Culture
At 42 h IVM, a cumulus-free oocyte was held with a holding micropipette (110-µm 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 µg/ml cytochalasin B (Sigma-Aldrich Corp.). After enucleation, oocytes were stained with 5 µg/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 µsec 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 acid-free BSA (Sigma-Aldrich Corp.), and placed in 25 µl microdrops (5–7 oocytes per drop) of NCSU-23 under mineral oil and cultured at 39°C, 5% CO2, 7% O2, and 88% N2. The reconstructed embryos were cultured for 7 days after activation.
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 µl 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.
Detection of EGFP Gene in Cloned Piglets
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, 5'-GCG ATG CCA CCT ACG GCA AGC TGA-3, and antisense, 3'-GAG CTG CAC GCT GCC GTC CTC GAT-5'. The genomic DNA (300 ng) was amplified in a 50-µl PCR reaction containing 2.5 units Hotstart Taq polymerase (Qiagen, Hilden, Gemany) 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 94°C, annealing for 35 sec at 58°C, extension for 90 sec at 72°C, and a final extension for 15 min at 72°C. 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 µM calcium ionomycin for 4 min (G-EFIA and S-EFIA groups, respectively), or 3) were subjected to electrical fusion and subsequent activation by 15 µM 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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).
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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).
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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 (-)] 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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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].
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Betthauser et al. [4] reported that delayed activation with ionomycin + 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 
-1,3-galactosyltransferase 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 allocations 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 cell-cycle 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 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 -1,3-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 GFP-expressing 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, Kühholzer 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 39°C, 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 surrogates 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Woosuk Hwang, Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151–742, Korea. FAX: 822 884 1902; hwangws{at}snu.ac.kr
Received: 19 December 2002.
First decision: 20 January 2003.
Accepted: 8 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-1,3-galactosyltransferase knockout piglets by nuclear transfer cloning. Science 2002 295:1089-1092This article has been cited by other articles:
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  Y.-M. Zheng, H.-Y. Zhao, X.-E Zhao, F.-S. Quan, S. Hua, X.-Y. He, J. Liu, X.-N. He, and H. Lin Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells transfected with enhanced green fluorescence protein gene Reproduction, May 1, 2009; 137(5): 793 - 801. [Abstract] [Full Text] [PDF]
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 X. J. Yin, H. S. Lee, X. F. Yu, E. Choi, B. C. Koo, M. S. Kwon, Y. S. Lee, S. J. Cho, G. Z. Jin, L. H. Kim, et al. Generation of Cloned Transgenic Cats Expressing Red Fluorescence Protein Biol Reprod, March 1, 2008; 78(3): 425 - 431. [Abstract] [Full Text] [PDF]
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