Nuclear transfer (NT) technology can be used to clone desirable adult genotypes and phenotypes and, thus, is a valuable tool for agriculture and biomedical purposes (Schnieke et al.,1997; Lai et al.,2002; Wilmut and Paterson,2003). An important advancement was made in the field recently when several research groups succeeded in cloning piglets from somatic cells (Betthauser et al.,2000; Onishi et al.,2000; Polejaeva et al.,2000; Bondioli et al.,2001; Yin et al.,2002,2003; Park et al.,2004,2005). This finding has permitted the genetic improvement of farmed pigs and the production of transgenic pigs for medical use and organ transplantation (Lai et al.,2002).
Using transfected somatic cells as the karyoplast donors in NT can generate transgenic cloned animals. Compared with the alternative approach, namely, microinjecting the gene into the embryo, the use of transfected somatic cells as the karyoplast donor is associated with several practical advantages, as follows (Wilmut and Paterson,2003). First, the donor cells can be prescreened for desirable genotypic characteristics, which can reduce the number of donor and recipient animals needed for the procedure. This is useful because, on the basis of the protein production characteristics of the transgenic animals, it is possible to predict the protein production characteristics of the cloned progeny. Second, dead transgenic animals can be restored by using them as donor cell source to reproduce transgenic animals.
Despite the advantages of somatic cell NT (scNT), this procedure produces normal offspring with a very low efficiency. This finding is in part because of a high incidence of fetal death that is due to a variety of afflictions during pregnancy, including fetal overgrowth and placental malformations (Hiendleder et al.,2004; Arnold et al.,2006; Chae et al.,2006; Lee et al.,2007). These studies have revealed that some of the subtle abnormalities of adult scNT-derived animals are species-specific. It should be noted, however, that some of the phenotypic effects of scNT cloning, such as increased size and mass, polydactyly, swollen edematous skin, and perinatal death, have also been observed in embryonic stem cell-derived animals (Dean et al.,1998). However, compared with other reproductive assistant techniques, scNT cloning is associated with particularly high levels of phenotypic instability (Hill et al.,1999; Cibelli et al.,2002; Hiendleder et al.,2004; Park et al.,2004,2005), as 64% of cattle, 40% of sheep, 60% of pigs, and 93% of mice exhibit some form of abnormality, such as pulmonary hypertension and respiratory distress. For example, goat and pig clones are prone to bacterial infections of the lungs, abnormal numbers of teats, cleft lips, malformed limbs, an atrial septal defect, a cranial abnormality, and contracted tendons (Carter et al.,2002; Piedrahita et al.,2004; Lee et al.,2005). It should also be noted that many researchers have observed that cloned animals are healthy and normal (Bondioli et al.,2001; Lanza et al.,2001; Lai et al.,2002; Yin et al.,2002). Nevertheless, the usefulness of scNT is clearly reduced by its low cloning efficiency and the high levels of phenotypic instability that has been observed in cloned animals that survive to adulthood.
The unpredictable developmental potential and phenotypic outcome of scNT-derived clones is probably in part because scNT involves a de-differentiation process (Wakayama et al.,2000; Wilmut et al.,2002; Kubota et al.,2004). The scNT-derived organisms must be built from a fusion between an enucleated oocyte and a fully differentiated cell, and inadequate de-differentiation of the latter would certainly lead to many developmental problems: many clones also fail at early stages of development simply because of physical damage to the nucleus or the cell's viscous innards, which are full of chemicals that are essential for normal development (Humpherys et al.,2002). A growing number of studies have also suggested that even those scNT-derived clones that survive to adulthood might bear subtle genetic abnormalities that could cause medical problems later. To determine the scope and consequences of this problem, many cloned animals have been closely monitored until they die. Thus, insufficient reprogramming of the somatic cell nucleus due to unreliable NT techniques may be responsible for much of the phenotypic instability associated with scNT. However, it is also possible that inherent or accidental genomic damage of the donor cells may also be responsible for the phenotypic instability of the offspring and the low efficiency of scNT (Mastromonaco et al.,2006; King et al.,2006). We here tested whether scNT cloning-associated abnormalities are due to inherent/accidental genomic damage of the donor cells or faulty epigenetic reprogramming. To prove our hypothesis, we produced serial second-generation (G2) and third-generation (G3) clones from an abnormal first-generation (G1) clone and found that all four G2 clones and two G3 clones showed a normal outward appearance, indicating that the abnormality of the G1 clone may be caused by epigenetic reprogramming of only a few of the clone's cells during development.
Generation of scNT-derived Transgenic Pigs
An expression vector containing the full-length human thrombopoietin (hTPO) genomic DNA was constructed by a combination of human Lambda FixII genomic library screening and polymerase chain reaction (PCR) amplification. An expression vector with the truncated genomic hTPO gene (plus an artificial stop codon) was constructed by PCR. The truncated and full-length hTPO fragments, which both contain erythropoietin (EPO) -like domains and an 18 amino acid region containing two O-glycosylation sites, were ligated into an expression vector driven by the pig uroplakin II (pUPII) promoter (Fig. 1A,B). The expression of the full-length (332 amino acids; ∼70 kDa) and truncated (171 amino acids; ∼18 kDa) hTPO proteins by transfected RT4 cells was confirmed by Western blot analysis (Fig. 1C).
The truncated hTPO expression vector was transfected into porcine fibroblast cells (Landrace x Duroc F1) and selected by G418 treatment for 3 weeks. The transfected cells were then used as donor cells (D0) in scNT. A total of 2,818 scNT embryos were transferred to 11 recipients and a total of five G1 piglets were obtained (Table 1). However, two of these transgenic piglets died within 7 days of birth. Southern blot analysis revealed that each clone contained at least four copies of the transgene (Fig. 2A). The #183-1 G1 transgenic clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge (Fig. 3A). The other clones looked normal. The chromosomal localization of the transgene insertion site in the #183-1 clone was then determined by fluorescent in situ hybridization (FISH) using a full genomic DNA as a probe and a signal from the transgene was detected in chromosome 8p1.2 (Fig. 2B–D). The same result was obtained with another transgenic pig (#183-2) that had a normal appearance (data not shown). Thus, the expression of the exogenous hTPO gene was not responsible for the phenotypic abnormality of #183-1.
Table 1. Efficacy of Serial scNT Cloning Starting With Truncated hTPO Gene-Transgenic Somatic Cellsa
Clones (Donor cell generation)
No. of embryos transferred
No. of recipients
No. pregnant (%)
No. of piglets
Efficiency per embryo (%)
Gestation length (days)
Values that have different superscript numbers differ significantly (P < 0.05). G1, G2, and G3 indicate the first, second, and third generations of scNT pigs, respectively. D0, D1, and D2 indicate the donor cells used to generate the first, second, and third generations of scNT pigs, respectively. D0 cells were derived from gestational day 30 fetuses, whereas the D1 and D2 donor cells were derived from ear cells of G1 and G2 pigs. scNT, Somatic cell nuclear transfer; hTPO, human thrombopoietin.
118.5 ± 2.12
120.0 ± 3.61
123.5 ± 6.36
Phenotypic Abnormalities of scNT Pig May Be Induced by Reprogramming Error Rather Than DNA Damage
To determine whether the phenotypic abnormalities of scNT cloned animals are caused by inherent/accidental genomic damage of the donor cells or by faulty epigenetic reprogramming, we serially cloned #183-1 by using its ear-derived fibroblast cells in scNT and assessed whether its G2 and G3 descendants also had its facial abnormality. All four of the G2 clones that were obtained (Table 1) had a normal outward appearance (Fig. 3A), which indicates that the phenotypic instability of the G1 clone was not due to genomic damage of the donor cell. If the phenotypic abnormality of the #183-1 clone reappeared in the G3 clones, epigenetic dysregulation in a specific region rather than over genome-wide regions would be the most likely explanation. However, the two G3 clones obtained (Table 1) were normal in appearance (Fig. 3A).
To confirm that the piglets were identical to the donor cell line used, parentage analysis was performed using DNA obtained from ear punches of the scNT piglets and the surrogate recipients (Fig. 3B). Southern blot analysis of three porcine DNA microsatellite (MS) markers (SWR1120, SW1311, and SW1327) clearly showed that the recloned pigs (G2 and G3 clones) were genotypically identical and that the donor cells were the source of the genetic material used to produce the newborn piglets.
To examine whether phenotypic abnormalities in scNT-derived clones are induced by aberrant epigenetic reprogramming of fetal fibroblasts or donor cell damage, we analyzed DNA damage of donor cells (Fig. 4) and the global methylation changes that take place during the development of preimplantation G1 and G2 embryos (Fig. 5). The Comet assay revealed that the Comet tail moment of scNT-derived donor cells (D1, D2, and D3) was not statistically different from that of normal donor cell (D0), although significantly different from H2O2-treated control cells, strongly supporting that scNT-derived donor cells might be as normal as D0 and the phenotypic abnormalities of scNT cloned animals might be not caused by inherent/accidental genomic damage of the donor cells. Furthermore, the methylation patterns in the PRE-SINE repeats sequences of the G1 clone (94/140, 67.18 ± 1.63%) were similar to those in the G2 clones (147/207, 72.06 ± 2.61%). Furthermore, we could not find any significant differences in the methylation patterns between the G1 and G2 clones (Fig. 5). Collectively, these results suggest that the abnormality of the G1 clone may have been caused by the impaired epigenetic reprogramming of just a few of the clone's cells during gestational development rather than by genomic damage or genome-wide reprogramming errors.
Low Efficacy of the scNT Cloning May Be Caused by Abnormal Placenta Development Rather Than Abnormal Cumulative Genomic Damage
We assessed the efficacy of the serial cloning in terms of the number of transferred embryos that survived to term. Of the NT eggs at the one- to four-cell stage that were surgically transferred to the oviducts of recipient gilts, 0.18% of the G1 embryos (1/564) survived until birth, whereas only 0.042% (1/2,409) and 0.035% (1/2,849) of the G2 and G3 clones survived until birth, respectively (Table 1). Thus, the second and third rounds of cloning were significantly less efficient than the first round (P < 0.05). To determine whether this difference was due to impaired early development, we analyzed the developmental potentials of the G1, G2, and G3 scNT-derived preimplantation embryos (Table 2). G4 embryos were also examined. After electroactivation and cultivation in four-well dishes, 55.8% (134/240), 56.6% (136/240), 53.3% (128/240), and 51.6% (124/240) of the G1, G2, G3, and G4 oocytes underwent cleavage on day 2, respectively. After 7 days, 15.4% (37/240), 10% (24/240), 12.5% (30/240), and 13.3% (32/240) of the embryos cultured in the NCSU-23 medium developed to the blastocyst stage, respectively. Moreover, the cell numbers (30.9 ± 2.5) in G1-derived blastocyst stages were statistically similar to those in the other three cell generations (28.9 ± 3.0 for G2; 26.6 ± 3.1 for G3; 28.3 ± 3.7 for G4; Table 2). In addition, a significant change in the apoptotic cell index was not observed (Table 2). Thus, cumulative genomic damage does not seem to be responsible for the low efficacy of the scNT cloning.
Table 2. In Vitro Developmental Efficacy of Serial scNT-Derived Preimplantation Embryosa
Clones (donor cell generation)
No. of embryos
No. of embryos (%)
No. of embryos developing into blastocysts (%)
Cell no. in blastocysts
No. of apoptotic cells
G1, G2, G3, and G4 indicate the first, second, third, and fourth generation of scNT cloned embryos, respectively. scNT, somatic cell nuclear transfer.
30.9 ± 2.5
2.1 ± 0.6
28.9 ± 3.0
2.2 ± 0.6
26.6 ± 3.1
2.4 ± 0.6
28.3 ± 3.7
2.3 ± 0.9
To assess whether the hTPO transgene affected the serial cloning, we conducted another round of serial cloning, this time using somatic cells transfected with the human EPO (hEPO) gene (Table 3). The G1 generation of hEPO-transgenic embryos was produced with similar efficacy (1/525 embryos, 0.19%) as the G1 generation of hTPO-transgenic embryos (0.18%). As with the hTPO transgenic embryos, the efficacy in the hEPO G2 generation was lower compared with the first hEPO generation (0.11% vs. 0.19%), although this difference did not achieve statistical significance. However, the efficacy in the hEPO G2 generation was higher than that in the hTPO G2 generation (0.042%). Even though physiological conditions may also affect cloning efficiency, the transgene may affect the cloning efficacy to some extent.
Table 3. Efficacy of Serial scNT Cloning Starting With hEPO Gene-Transgenic Somatic Cellsa
Clone (donor cell) generation
No. of embryos transferred
No. of recipients
No. pregnant (%)
No. of piglets
Efficiency per embryo (%)
Gestation length (days)
G1 and G2 indicate the first and second generation of scNT pigs, respectively. Fetal fibroblast cells were derived from gestational day 30 fetuses and transfected with the hEPO gene under the control of the goat beta casein gene promoter (pBC1hEPO: for details, see Kwon et al,2006b). Cells carrying the pBC1hEPO gene were selected with G418 for 3 weeks and used as donor cells in scNT (D0). The D1 donor cells were derived from ear cells of G1 pigs. scNT, somatic cell nuclear transfer; hEPO, human erythropoietin.
121.0 ± 1.41
121.8 ± 2.64
Regardless of the generation involved, the hTPO and hEPO cloning efficacy was very low. Thus, we next examined whether the low efficacy of scNT cloning was related to placenta defects. For this, the term-derived placentas from immediate-postpartum hTPO scNT and control pigs were compared. The scNT placentas had severely damaged cytotrophoblasts, but their syncytiotrophoblasts were less seriously damaged (data not shown). Flow cytometric analysis of the cell populations in the term placentas revealed the similar levels of uPA-, uPAR-, Met-, Grb-, and Bek-positive cells but higher numbers of Ets-1–, SOS-1/2–, and fibroblast growth factor receptor-3 (FGFR-3) -positive cells in the control placentas (Table 4). The control placentas also had fewer Erk1/2-positive cells and Ras and matrix metalloproteinase-9 (MMP-9) -positive cells were not found in scNT placenta (Table 4). Immunohistochemical analysis also revealed that scNT-derived term placentas showed extensive Sos-2 staining in the cytotrophoblast and syncytiotrophoblast cells, which are located along the basement membrane layer of branched villi, whereas in the control placentas, Sos-2 protein was abundantly detected in the cell column of the anchoring villus (Fig. 6). Collectively, our data demonstrate that the metabolic patterns in the fetoplacental unit in developing scNT fetuses may be altered and that there is loss of surface area. These changes have been associated with a poor maternofetal exchange capacity and intrauterine growth restriction. Thus, the low efficacy of scNT pig production may be due to placental insufficiency during gestation.
Table 4. FACS Analysis of Placenta Derived From Normal and scNT-Derived Pigs With Trophoblast-Specific Antibodiesa
% Positive cells by FACS
Values in rows that have different superscript numbers differ significantly (P < 0.05). N.D., not detectable; FACS, fluorescence-activated cell sorting; scNT, somatic cell nuclear transfer.
20.21 ± 2.2
17.8 ± 4.27
24.27 ± 8.1
18.56 ± 5.5
2.98 ± 2.1
17.6 ± 4.31
6.2 ± 0.962
29.23 ± 16.6
21.12 ± 4.97
38.27 ± 9.71
17.27 ± 3.82
24.47 ± 2.61
15.8 ± 5.62
5.3 ± 1.9
6.1 ± 2.42
11.84 ± 3.132
19.21 ± 7.3
28.22 ± 2.63
5.14 ± 2.15
6.06 ± 1.7
3.55 ± 2.6
pUPII Promoter Directs Specifically hTPO Gene Expression Into Urothelium of Ureter and Bladder
We also assessed the expression of the truncated hTPO gene in the six G2 and G3 transgenic pigs that we obtained from serial scNT from the G1 transgenic pig. As illustrated in Figure 7A, hTPO mRNA was detected mainly in the bladder and urethra of the transgenic pigs, although trace amounts of the transgene were also found in the spleen. Immunohistochemical staining for hTPO on formalin-fixed tissue sections revealed the truncated hTPO expression in the transgenic pigs was principally limited to the suprabasal cell layers of the urothelium in the bladder and the epithelium of the urethra (Fig. 7B). Furthermore, Western blot analysis of the urine of transgenic piglets revealed positive 18-kDa signals, although none of the adult transgenic pigs showed a positive signal (Fig. 8).
To investigate the side-effects of hTPO expression, four major blood parameters were examined, namely, platelet numbers, red blood cell numbers, hemoglobin content, and hematocrit. The transgenic pigs did not exhibit any apparent major changes in these blood parameters compared with the controls (data not shown). The lack of side effects in the transgenic pigs may be due to the low levels of hTPO expression or the tissue- and cell-specific expression in the transgenic tissues. In addition, the serially cloned pigs showed normal reproductive behavior and fertility.
The data presented here were collected from a study that spanned more than 3 yr and involved a large number of controls and cloned animals. During this study, we successfully produced 65 cloned piglets from 24 recipients using nine different donor cells (data not shown). Compared with artificial insemination (AI), scNT was associated with significantly lower pregnancy (57/265, 21.5%) and abortion (33/57, 57.8%) rates (unpublished data). The 65 scNT piglets were obtained from 265 recipients receiving 62,009 embryos. Of these, 14 piglets (21.5%) were stillborn, 38 scNT piglets (58.5%) died suddenly within the first week of life, and 3 piglets (4.6%) died during puberty. To date, only 10 scNT pigs (15.4%) are still alive and healthy. The scNT clones showed a higher incidence of postnatal death compared with the AI-derived piglets (5/144, 4.4%; three were stillborn and two were crushed). Histological analyses of 28 of the 38 scNT clones that died after birth revealed 11 had distinct histological anomalies, the most common of which was meningitis, followed by poor development of the elbow joint bone, flexor tendons, congestion, and Leydig cell hypoplasia (Park et al.,2004,2005). As shown in our very recent report, the abnormal histopathologic findings of the deceased cloned pigs are closely associated with aberrant protein expression patterns (Park et al.,2005; Chae et al.,2006; Lee et al.,2007).
At present, many scientists have been trying to find cause(s) of phenotypic abnormalities of scNT-derived offsprings and one of the possible causes could be explained by an oocyte source as a karyoplast donor for nuclear transfer. In general, in vitro matured ooctes are used for the production of transgenic pigs derived from scNT and would be expected to be less efficient and productive. Therefore, in vivo matured oocytes would be a better source for the karyoplast donor to produce healthy offspring, but nevertheless, they are limited to provide sufficient numbers of donor cells due to extremely low efficiency of scNT rates.
Previous studies have reported that scNT-derived clones are prone to several abnormal phenotypes, including respiratory distress (Garry et al.,1996; Walker et al.,1996; Carter et al.,2002), contracted tendons and cardiovascular problems (Schnieke et al.,1997; Hill et al.,1999; Carter et al.,2002; Lai et al.,2002), and large birth weight (Walker et al.,1996; Boquest et al.,2002). We and authors of another study reported that phenotypic instability in swine resulted from scNT (Lai et al.,2002; Archer et al.,2003; Park et al.,2004,2005). However, other groups have observed few problems (Betthauser et al.,2000; De Sousa et al.,2002; Walker et al.,2002; Yin et al.,2002). In cattle, the donor cell type was found to be responsible for some of the abnormal phenotypes that were observed, as cells derived from adults resulted in more abnormalities (Heyman et al.,2002). In our study here, one of the five G1 scNT piglet clones exhibited an abnormal nose phenotype. Because the five G1 piglets were not generated from donor cells derived from a cloned cell, our data suggest that phenotypic trait variability may be due to variable abnormalities in the fibroblast donor cells. To test this possibility, we produced G2 clones using somatic cells established from the G1 clone with the nose phenotype abnormality. Because the G2 clones were derived from the Gl clone, any abnormalities in these progeny would be expected to be a consequence of genomic damage in the D0 donor cells. However, if the progeny were to show a normal phenotype, then the phenotype abnormality of the G1 clone would be the consequence of aberrant epigenetic reprogramming. The birth weights of the two surviving G2 clones (0.8 and 1.2 kg) were slightly lower or equivalent to that of the G1 donor clone (1.2 kg), but all G2 clones had a normal outward appearance (Fig. 3A).
To reconfirm the relationship between phenotype instability and epigenetic hotspot(s) for aberrant DNA methylation, we made serial G3 clones from the fibroblast cells of a G2 clone. If the phenotypic abnormality of the G1 clone were to reappear in G3 cloned pigs, epigenetic dysregulation in a specific region rather than in genome-wide regions would be the most likely explanation. The birth weights of the two surviving G3 clones (1.4 kg) were equivalent to that of the donor G2 clone (1.2 kg) and both G3 clones had a normal appearance (Fig. 3A). Taken together, the phenotypic instability of the G1 clone cannot be adequately explained by the “genomic damage of the donor cell” hypothesis and may have resulted from epigenetic dysregulation.
Placental abnormalities in humans have been associated with adverse effects on the growth and development of the fetus (Sakai et al.,2005). Moreover, it has been shown that, although clones show a high incidence of phenotypic abnormalities such as obesity (Tamashiro et al.,2002) and premature death (Ogonuli et al.,2002) that are thought to arise from placental problems, the progeny of these clones that are generated by natural mating do not have these abnormalities. This finding suggests that only germ-line passage can completely reprogram the placental changes induced by cloning. To examine this issue, we investigated the term placentas of the scNT-derived clones (Table 4). Unlike the placentas of mice and cattle, the pig scNT clones had significantly lower term placenta weights (0.24 ± 0.04 kg; n = 6) and ponderal index (0.882 ± 0.243; n = 9) compared with control age- and sex-matched piglets (0.33 ± 0.08 kg and 1.182 ± 0.099; P < 0.05). Moreover, the scNT placentas showed a shallow floating villus, unlike the control placentas. They also had a more irregular and dystrophic shape and showed distinct histological defects indicating placenta insufficiency (Fig. 6). These anatomical defects suggest that scNT-derived cytotrophoblast differentiation along the invasive pathway was abnormal.
Recently, we reported that increased oxygen demand during embryonic development leads to increased rates of reactive oxygen species (ROS) production in the extraembryonic tissue of the placenta. ROS levels increase gradually during early pregnancy, resulting in the apoptotic cell death of extraembryonic tissues (Chae et al.,2006). Very recently, we also reported that term placenta malformation was caused by apoptosis of placenta cells by means of Bad up-regulation and down-regulation of 14-3-3 protein expression (Lee et al.,2007). In this study, scNT term placentas had significantly reduced expression of Ets-1, SOS-1/2, FGFR-3, MMP-9, and Ras, which are under the control of the Met/VEGF signal (Table 4; Munoz-Chapuli et al.,2004). A constitutively active form of Met, Tpr-Met, activates the uPA gene promoter through a Grb2/SoS/Ras/ERK cascade (Besser et al.,1997). Even though their transduction pathways in placenta are not well known, Ras/ERK pathway may be also involved in the up-regulation of MMP-9, which promotes uterus invasion of trophoblast. In addition, Sos-2 immunoreactivity was abundant in the cytotrophoblast and syncytiotrophoblast cells located along the basement membrane layer of branched villi (Fig. 6). This finding was rarely observed in the control placentas. Moreover, in normal but not scNT placentas, Sos-2 protein was abundantly detected in the cell column of the anchoring villus. This finding indicates that scNT-derived placenta cells fail to switch on the expression of stage-specific cells. Taken together, these observations suggest that placental insufficiency and the altered expression of trophoblast cells may be principally responsible for the high rates of embryonic loss and/or developmental abnormality (particularly during gestation) that are associated with scNT cloning.
The bladder is an attractive organ for the production of pharmaceutical proteins because urine can be easily collected during the lifetime of transgenic animals, irrespective of their sex (Kerr et al.,1998; Ryoo et al.,2001). It was previously shown that a 3.6-kb 5′ flanking region of the mouse uroplakin II (mUPII) gene could be used to induce the secretion of a pharmaceutical protein into the urine of transgenic mice (Kerr et al.,1998). The concentration of the mUPII/human growth hormone reached 0.1–0.5 μg/l in mouse urine, which represented 0.02% of total urinary protein in transgenic mice. However, studies of transgenic mice showed that the exogenous gene under the control of the 3.6-kb mUPII promoter gene is also expressed in tissues other than the bladder (Meyer-Puttlitz et al.,1995). These results suggest that it is preferable to use long upstream regulatory elements for bladder-specific gene expression. In this study, transgenic pigs harboring truncated hTPO genomic DNA (18 kDa) under the control of the 8.8 kb of pUPII promoter were produced. Transgene RNA was expressed exclusively in the bladder and ureter of the four transgenic pigs examined, and a trace amount of the transgene was also found in the spleen of one pig. In addition, we found that secreted hTPO protein was detectable in transgenic piglet-derived urine, not in adult urine, suggesting that failure of detection of secreted hTPO protein in adult urine might be due to the low levels of hTPO expression. Therefore, as a piglet grows to be adult, a low level of hTPO expression might be diluted in the increased amount of total urine and fail to be detected by Western blot analysis. Further study is required to analyze the expression level of hTPO protein at different developmental stages by increasing the number of breeding pigs. However, the transgenic pigs were normal, and a positive correlation between the amount of hTPO in the urine of the adult pigs and their blood platelet levels was not observed. On the basis of these results, we anticipate that the 8.8-kb fragment containing the pUPII promoter will efficiently direct hTPO expression in the bladder and ureter of transgenic pigs.
In conclusion, all of the recloned pigs described here did not exhibit phenotypic abnormalities, and the abnormalities we observed in the G1 generation may not have been due to genomic damage of the donor cell. Rather, the abnormal outward appearance, and perhaps other aberrant phenotypes as well, may have been the result of epigenetic alterations that presumably occurred during the cloning process itself and/or placenta insufficiency. Although G3 clones in mice have been analyzed (Wakayama et al.,2000), to our knowledge, this study represents the first analysis of the performance of third-generation clones of livestock clones.
Animal care and experiments were conducted in accordance with the Kon-Kuk University Guideline for the Care and Use of Laboratory Animals.
Cloning of Full-length and Truncated Human thrombopoietin (hTPO) Genomic DNA
To avoid point mutations or deletions, we obtained the hTPO gene by human library screening (Stratagene, La Jolla, CA) as described previously (Kwon et al.,2002). The probe used for the screening was generated as follows. The genomic hTPO gene sequence was obtained from GenBank (accession no. L36052), and this sequence was used to generate the following PCR primers: forward 5′-CTCCTCCTGCTTGTGACCTC-3′ and reverse 5′-ATGTCCTGTGCCTTGGTCTC-3′. The hTPO probe PCR product (181 bp) was then cloned into the pGEM T-easy vector (Promega, Madison, WI). DNA sequencing confirmed that the DNA fragment had the sequence of genomic hTPO DNA. Approximately 1.0 × 107 plaques from a human Lambda FixII genomic library (Stratagene) were screened for the hTPO genomic fragment, and four clones were obtained. However, the obtained fragments (8 kb) only contained part of the hTPO open reading frame, namely, a sequence that extended from the 5′-UTR to intron 5 and included exon 5. Thus, we amplified a 1.5-kb fragment that consisted of exon 6 through to the stop codon. For this, we used the 5′-GGATCCCAATGCCATCTTCCTGAG -3′ primer, which contains a BamH1 site downstream (5,169 bp) of exon 6, and the 5′-AAGCTTATTCATTTCTCAAG-3′ primer, which encodes the stop sequence. The full-length genomic hTPO gene (5.4 kb, 70 kDa) was then constructed by recombination of both fragments.
To construct the truncated hTPO (4.5 kb [683-5318], 18 kDa), hTPO genomic DNA that contained four exons and two O-glycosylation sites was digested with NotI and XhoI enzymes. Another hTPO genomic DNA fragment that was approximately 600 bp in size and contained an artificial stop codon and an XhoI enzyme site was amplified by PCR, cloned into pSK (+), and sequenced for point mutations. Both fragments were then digested with SalI and XhoI and ligated into the pSK (+) vector (Stratagene).
Construction of hTPO Expression Vectors Under the Control of the 8.8-kb pUPII Promoter
Approximately 8.8 kb of the pUPII promoter was isolated from a pig Lambda Fix II genomic library (Stratagene) as described in our previous report (Kwon et al.,2006). The expression vector pBC1 (Invitrogen, Carlsbad, CA) was completely digested with BamHI and NotI to remove the casein promoter DNA and the poly A site and then ligated with a 8.8-kb fragment containing the pUPII promoter to create a pUPII expression vector (Kwon et al.,2006a,b). To construct hTPO expression vectors, the full-length (5.4 kb) or truncated hTPO genomic DNA (4.5 kb) was ligated with a 2.7-kb fragment containing SV 40 poly A. In addition, a PCR-amplified woodchuck hepatitis virus posttranscriptional regulatory element (WPRE fragment; nucleotide 1093 to 1684; GenBank accession no. J04514) was completely digested with XhoI. The resulting WPRE fragments were inserted into the unique XhoI site located downstream of the hTPO stop codon. The 8.1- and 7.2-kb DNA fragments containing hTPO and SV40 poly A were isolated from pSK (+) by digestion with AscI and NotI and then ligated into the pUPII expression vector. As shown in Figure 1, the resulting 19- or 20-kb bladder-specific expression vector containing full-length or truncated genomic hTPO was isolated by SalI digestion and was then used for somatic cell transfection.
Preparation of Donor Cells
D0 donor cells were derived from a gestational day 30 fetus of a Duroc × Landrace sow. Fibroblasts of G1–G3 clones were obtained from the ears at one day postpartum and were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) under 5% CO2 at 37°C. To generate G1 clones that stably expressed the TPO gene, the donor cells were used at passages 7–9. G2–G4 clones were produced by using donor cells at passages 2–3. Donor cells D0, D1, D2, and D3 were also frozen for further analyses of embryo development in vitro.
DNA damage in donor cells was measured by using CometAssay kit according to the manufacturer's protocol (Trevigen, Gaithersburg, MD). Cells were randomly analyzed per slide and scored for Comet tail moment. H2O2-treated cells were used for a control. Comet Assay data were analyzed using Comet Assay IV software program (Version 4.2, Perceptive Instruments Ltd., Haverhill, Suffolk, UK).
D0 cells were grown on 24-well plates until they were 50–70% confluent and then were transfected for 2 hr in serum- and antibiotic-free DMEM with 1 μg/well pUPII/truncated hTPO plasmid that had been complexed with the cationic commercial liposome Geneporter2 (Qiagen, Valencia, CA). The liposomes were used at the ratio recommended by the supplier (DNA:liposome = 1:4, w/w). The cells were subsequently thoroughly washed with DMEM plus 10% FBS, cultured for another 2–3 days until they were confluent, and then selected with G418 for 3 weeks.
In Vitro Maturation of Oocytes
Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory at 25°C–35°C. Antral follicles (2–6 mm in diameter) were aspirated with an 18-gauge needle. Aspirated oocytes that had an evenly granulated cytoplasm and were surrounded by at least three uniform layers of compact cumulus cells were selected and washed three times in Hepes-buffered NCSU-37 with 0.1% polyvinyl alcohol (PVA). Oocytes were cultured for 20 hr in four-well plates (Nunc, Roskilde, Denmark), each well of which contained 500 μl of NCSU-37 medium supplemented with 10% porcine follicular fluid, 0.6 mmol/L cysteine, 1 mmol/L dibutyryl cyclic adenosine monophosphate (dbcAMP, Sigma, St. Louis, MO), and 0.1 IU/ml human menopausal gonadotropin (hMG, Teikokuzoki, Tokyo, Japan). The oocytes were then cultured without dbcAMP and hMG for another 18–24 hr as previously reported (Yin et al.,2002).
NT was carried out as described previously (Yin et al.,2003; Park et al.,2004,2005, Chae et al.,2006; Lee et al.,2007). Briefly, the matured eggs with the first polar body were cultured in NCSU-23 medium supplemented with 0.4 mg/ml demecolcine (Sigma) and 0.05 mol/L sucrose for 1 hr. The sucrose was used to enlarge the perivitelline space of the eggs. Treated eggs with a protruding membrane were moved to medium supplemented with 5 mg/ml cytochalasin B (CB) and 0.4 mg/ml demecolcine, and the protrusion was removed with a beveled pipette. A single donor cell was injected into the perivitelline space of each egg and electrically fused by using two direct current pulses of 150 V/mm for 50 μsec in 0.28 mol/L mannitol supplemented with 0.1 mM MgSO4 and 0.01% PVA. The fused eggs were cultured in medium with 0.4 mg/ml colcemid for 1 hr before parthenogenetic activation, and then cultured in 5 mg/ml of CB-supplemented medium for 4 hr. The reconstructed oocytes were activated by 2 direct current pulses of 100 V/mm for 20 msec in 0.28 mol/L mannitol supplemented with 0.1 mmol/L MgSO4 and 0.05 mmol/L CaCl2 (Yin et al.,2003; Park et al.,2004,2005). To obtain simultaneously activated oocytes, a group of oocytes were fused and activated with two 50-μsec pulses of 1.5 kV/cm. The activated eggs were then cultured in NCSU-23 medium for 7 days in an atmosphere of 5% CO2 and 95% air at 39°C.
Estrus Synchronization and Embryo Transfer
Gilts (Duroc × Yorkshire) that were at least 8 months of age served as recipients. Estrus synchronization of the recipients was established as reported previously (Yin et al.,2003; Park et al.,2004,2005; Chae et al.,2006; Lee et al.,2007). One or two cells of scNT embryos were then surgically transferred into the oviducts of the synchronized recipients. The pregnancy status of the recipients was determined by ultrasound between days 25 and 30.
In Vitro Development of Serial Cloned Embryos
scNT was performed using donor cells after thawing passage 7∼9 of D0 and passage 2∼3 of D1–D3 cells, and reconstituted oocytes were activated as described above, then cultured in NCSU-23 medium for 7 days. Developmental efficacy and blastocyst formation was examined at the respective time point of cell stages up to 7 days. At the end of culture period, nuclei in blastocysts were counted after 5 μg/ml Hoechst staining.
Genomic DNA Isolation, Bisulfite Treatment, PCR Amplification, and Restriction Analysis
All DNA procedures have been described previously, as have the satellite region primer and PRE-1 primer sequences that were used (Kang et al.,2001; Yin et al.,2003; Park et al.,2004). In brief, somatic cells (1,000–3,000 in number) and scNT-derived NT blastocysts (20–30 in number) obtained by in vitro culture were subjected to bisulfite treatment. Cells were solubilized in 100 μl of lysis buffer containing 200 μg/ml of proteinase K, then incubated at 55°C for 3–5 hr. Genomic DNA was recovered from the lysate by ethanol precipitation in the presence of 5 μg of yeast tRNA as a carrier, then redissolved in 10 μl of distilled water. The genomic DNA was digested with BamHI enzyme in 20 μl of reaction volume for 16 hr, then denatured with 0.3 N NaOH. Bisulfite modification was initiated by adding 235 μl of freshly made 5 M sodium bisulfite (pH 5, Sigma) and 13.5 μl of 10 mM hydroquinone. The reaction mixture was covered with mineral oil and incubated at 55°C for 16 hr in the dark. The sample was removed from the mineral oil, and the bisulfite-treated genomic DNA was recovered using a Prep-A-Gene DNA purification kit (Bio-Rad, Hercules, CA). Desulfonation was performed by adding a one-tenth volume of 3 N NaOH and incubating at 37°C for 30 min. After precipitation, the DNA was resuspended in 20 μl of distilled water. For amplification of the pig PRE-1 sequence (GenBank Z75640), PCR was performed three times, each time with 3 μl of the bisulfite-converted genomic DNA as a template. The primer set used was 5′-TTTGTAGAATGTAGTTTTTAGAAG-3′ and 5-′AAAATCTAAACTACCTCTAACTC-3′. The amplification cycle was 45 cycles at 94°C for 60 sec, 55°C for 60 sec, 72°C for 20 sec, then finishing with one cycle of 72°C for 10 min. The PCR products pooled from three independent amplifications of the satellite DNA were cloned into TA-cloning vector (Promega, Madison, WI). Individual clones were sequenced using an automatic sequencer (ABI PRISM 377). Sequence analysis confirmed that bisulfite treatment was efficient enough to completely convert base Cs into Ts and the percentage of methylation were calculated as the ratio of converting Cs to Ts in donor cells and blastocysts. For TaqI restriction digestion of the PRE-1 sequences, a tenth volume of pooled PCR products was reamplified under the same conditions in the presence of 2 μCi P32-dCTP. Labeled PCR products were purified and concentrated using a Wizard DNA purification kit (Promega). One hundred nanograms of labeled DNA was digested with 20 units of TaqI restriction enzyme (Promega) overnight at 65°C, resolved on 5% polyacrylamide gel under nondenaturing conditions, dried, and exposed to autoradiographic film.
Parentage analysis was performed on the piglets obtained by scNT and the surrogate recipient females. DNA was extracted from the donor cells and from ear punches or tail clippings obtained from each recipient and newborn piglet. Three porcine DNA microsatellite markers (SWR1120, SW1311, and SW1327) were used to confirm the genetic identity of the cloned piglets as coming from the donor cells used for scNT (Yin et al.,2003; Park et al.,2004,2005).
Screening of Transgenic Pigs
Genomic DNA was obtained from pig ears, and the hTPO transgene was identified by PCR analysis. The following primers were used: forward hTPO primer, 5′-tcctgaaaaacctgtctggg-3′, and reverse hTPO primer, 5′-ttggactccaagtcctgaaatact-3′. Transgenic piglets detected by PCR analysis were reconfirmed by Southern blot analysis. For this, 10 μg of genomic DNA was digested with the AscI and NcoI restriction enzymes, electrophoresed in a 0.7% agarose gel, and transferred onto a nylon membrane. Hybridization was carried out in an aqueous solution containing 6× standard saline citrate (SSC), 5× Denhardt's reagent, and 1% sodium dodecyl sulfate (SDS) at 68°C using a random-primed full-length hTPO cDNA probe with 5× 106 cpm/ml activity. Final washes were performed with 1× SSC containing 0.1% SDS at 65°C.
Pig blood metaphase chromosome spreads were prepared by using a standard protocol for pig chromosomes (Kuipers et al.,1997; Wang et al.,2004). Porcine karyotyping was accomplished based on a proposed porcine chromosome nomenclature (Lin et al.,1980). FISH was performed by a standard method (Kuipers et al.,1997; Wang et al.,2004) using the entire transgene as a probe. Slides hybridized with a fluorescein isothiocyanate (FITC)-labeled probe were washed in 0.4× SSC at 73°C for 2 min and rinsed in 2× SSC/0.1% NP-40. Metaphase spreads were counterstained with 0.1 mg/ml propidium iodide. All slides were analyzed by a cooled CCD camera system attached to a fluorescence microscope with filters for FITC and Smart Capture software (CytoVision Chromofluor System, Applied Imaging Ltd, Carlsbad, CA). Two transgenic pigs with the transgene were examined. They had the same karyotypes.
Flow Cytometry Analysis
Flow cytometric analyses were performed as described previously (Choi et al.,2004). Briefly, term-placenta-derived cells was washed (5 × 106 cells per ml) in D-PBS, resuspended in staining buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 5 mM CaCl2) containing each antibody, and incubated for 30 min on ice. Antibodies specific for Met, Grb2, M-Ras, Ets-1, Bek, SOS-1, SOS-2, uPA, FGFR-3, Erk1/2, uPAR, MMP-9, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For indirect staining after incubation with primary monoclonal antibody, secondary FITC-conjugated antibodies (goat anti-rabbit IgG-FITC, goat anti-mouse IgG-FITC; Beckman Coulter, Fullerton, CA) were added for 30 min on ice, in darkness. Between the incubations, the cells were washed with ice-cold PBS supplemented with 2% fetal calf serum. Isotype-matched immunoglobulins (Beckman Coulter) were used as negative controls for nonspecific immunofluorescence. Flow cytometric analysis was performed at least three times with independent samples using Altra FACS (Beckman Coulter).
Terminal Deoxynucleotidyl Transferase–Mediated Deoxyuridinetriphosphate Nick End-Labeling Analysis
The terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) assay was performed as previously described (Choi et al.,2004,2006), with slight modifications. Blocks were subjected to immunohistochemical analysis using the TUNEL method (In Situ Cell Death Detection kit, Boehringer Mannheim, Germany).
Detection of the Truncated Human TPO mRNA and Protein Expression
Total RNA was isolated from various tissues (brain, kidney, liver, lung, heart, stomach, intestine, mammary gland, testis, ovary, bladder) and CHO (Chinese hamster ovary; negative control) cells maintained in Dulbecco's modified eagle's medium supplemented with 10% FBS, using the TRIzol reagent (Gibco BRL, Life Technologies, Carlsbad, CA). Northern blot analysis was performed as previously described (Kwon et al.,2002) using random-labeled human TPO cDNA.
Immunohistochemical staining was performed as described previously (Choi et al.,2004). Briefly, paraffin tissue sections were cleared in Histoclear for approximately 10 min and dehydrated in decreasing concentrations of ethanol. Immunohistochemistry was performed by using an ABC kit (Oncogene Science Inc., Boston, MA) according to the standard procedures provided by the manufacturer. Sections were placed in 3% peroxide in pure methanol and 0.1% pepsin in 0.05 N HCl (pH 2.25) for 30 min to reduce background staining. The sections were then washed twice (5 min each) in TBS (0.05 M Tris-HCl, pH 7.4, and 0.85% NaCl), blocked with normal horse serum (NHS) diluted in TBS (1:5; NHS-TBS), and incubated overnight with the primary anti-hTPO polyclonal antibody (Santa Cruz, CA) diluted at a concentration of 1:500 in NHS-TBS. One drop of NHS from the ABC Kit was used as a negative control. After removing excess antibody by two 5-min washes with TBS, the sections were incubated with biotinylated secondary IgG for 30 min, rinsed for 5 min 3 times with fresh TBS, incubated with ABC reagent for 30 min, washed extensively with TBS, and then rinsed in 1% Triton-X-PBS for 30 sec. The color reaction was developed by adding a solution of 0.5% diaminobenzidine in 0.05 M Tris-HCl (pH 7.6) containing 0.01% hydrogen peroxide. After developing the color reaction, the sections were washed in water, dehydrated, and mounted on glass slides with a coverslip.
All experimental data are presented as means ± SD. Each experiment was performed at least three times and subjected to statistical analysis. For statistical analysis (Tables 1–4), one-way analysis of variance was performed to determine whether there were significant differences among all groups (P < 0.05), and Fisher's post-test was performed to determine the significance of differences between pairs of groups. A P value below 0.05 was considered significant. Statistical tests were performed by using StatView software version 5.0 (SAS Institute Inc, Cary, NC).
This study was supported in part by the Research Project on Biogreen 21 from RDA, Republic of Korea.