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Influences of somatic donor cell sex on in vitro and in vivo embryo development following somatic cell nuclear transfer in pigs.

INTRODUCTION

The technique of somatic cell cloning in mammalians has been developed in the last few decades after the production of the first cloned sheep from a mammary gland cell [1]. Ever since the first somatic cell cloned pigs were produced by three different research groups [2-4], the technology of somatic cell cloning in animals has been applied to many fields, such as genetic improvement of farm animals, rescue of endangered species, and production of transgenic animals for bioscience research and agricultural purposes [5-7]. To date, porcine somatic cell cloning has been very difficult, with only 1% to 7% of the reconstructed embryos developing to full term [8]. The difficulty in porcine cloning has been attributed to multiple factors, including quality of recipient oocytes (in vivo vs in vitro matured), donor cell type, inadequate culture and manipulation media, oocyte activation method, requirement of a minimum number of fetuses, and adequate recipient conditions to maintain a pregnancy in the pig [9]. Several factors related to production of the cloned animals might affect the success rate of pig cloning, such as recipient breed [10], ovulation status of surrogate, in vitro culture time of the transferred cloned embryos [11-14], transferred cloned embryo number per surrogate [6,15], embryo transfer position [6], and embryo handling and transfer methods [16].

Previous studies have applied different approaches for improving pig cloning efficiency and proposed that the selection of a suitable donor cell type could increase the success rate of cloned piglets [15,17]. In cows, comparison of the efficiencies of various cell types from adult, newborn, and fetal male and female donor cells showed no significant difference in the percentages of blastocysts produced from each cell type [18]. Similar results have been attained using various cell types derived from different strains, sexes, and ages in mice [19]. Based on these studies, the use of donor cells from different origins was discovered to be one of the key factors affecting cloning efficiency and survival rates of cloned piglets. Therefore, effort must be undertaken to minimize inefficiencies at each step of the somatic cell cloning procedure. In previous research, the developmental competences of male and female somatic cell derived nuclear transferred embryos have not been adequately studied [20]. Therefore, the present study was conducted to investigate the developmental competence of somatic cell cloned porcine embryos derived from either a male or female fetal fibroblast cell as the donor cell.

MATERIAL AND METHODS

Chemicals and media

Unless otherwise stated, all chemicals, media and reagents used in the present study were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). All animal experiments were approved by and performed following the guidelines of the Pusan National University Animal Care and Experimentation Committee.

In vitro maturation of oocytes

Porcine ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to the laboratory at 30[degrees]C to 35[degrees]C. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles (3 to 6 mm in diameter) with an 18-gauge needle. COCs with evenly granulated cytoplasm and at least three uniform layers of compact cumulus cells were selected for further study and washed three times in HEPES-buffered North Carolina State University (NCSU-23) medium supplemented with 0.1% polyvinyl alcohol (PVA). The COCs were then cultured in 500 mL of maturation medium in four-well multidishes. The maturation medium was a modified NCSU-23 solution containing 10% (v/v) porcine follicular fluid, 0.6 mM cysteine, 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP), and 0.1 IU/mL human menopausal gonadotropin (Teikokuzoki, Tokyo, Japan). The maturation process was carried out for 20 h in the above medium at 38.5[degrees]C (with 5% CO2 and humidified air) and oocytes were subsequently cultured in the maturation medium without dbcAMP and hormones for another 18 to 24 h as previously described [21].

Isolation and culture of porcine somatic cells

Fibroblasts were isolated from pig fetuses on days 30 to 40 of gestation and the sex of the fetal fibroblasts were confirmed using polymerase chain reaction (Figure 1). The cells were cultured in Dulbecco's modified Eagles medium supplemented with 10% fetal bovine serum under 5% CO2, at 38.5[degrees]C in a humidified atmosphere. After reaching confluence, the cells were passaged. Donor cells were used for nuclear transfer between passages 4 and 10 of the culture, and the cells were used for nuclear transfer within 3 days of reaching confluence.

Somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) was performed as previously described [21]. Briefly, the matured eggs with the first polar body were cultured in medium supplemented with 0.4 mg/mL demecolcine and 0.05 mol/L sucrose for 1 h. 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 and 0.4 mg/mL demecolcine and the protrusion was removed using a beveled pipette as the micromanipulator. A single donor cell was injected into the perivitelline space of an enucleated oocyte and electrically fused using two direct current pulses of 150 V/mm for 50 [micro]s in 0.28 mol/L mannitol supplemented with 0.1 mM MgS[O.sub.4] and 0.01% PVA. Fused oocytes were then incubated in PZM5 medium containing 3 mg/mL fatty acid free for seven days with 5% C[O.sub.2] at 38.5[degrees]C in a humidified atmosphere, or for two days followed by transfer into the oviducts of recipient gilts. In the latter case, embryos were either harvested five days after transfer or allowed to develop to term.

Estrus synchronization

Estrus synchronization for preparation of recipients was carried out as previously described [3,21]. Briefly, an i.m. injection of 0.2 mg cloprostenol, a prostaglandin F2 alpha analogue (Planate; Sumitomo Seiyaku, Osaka, Japan), was administered to pregnant gilts (8 mo old, 120 to 130 kg) on days 33 to 53 of gestation, followed by a second injection of 0.2 mg cloprostenol 24 h later. One thousand international units of eCG (PMS 1000; Tani, NZ) was administrated i.m. at the same time as the second cloprostenol injection. Ovulation was induced by i.m. injection of 500 IU hCG (Puberogen; Sankyo, Tokyo, Japan) 72 h after the eCG injection. Ovulation was expected to occur 41 to 42 h after the hCG injection.

Embryo transfer

The nuclear-transferred eggs were activated with electric pulses and cultured for one or two days. One or two cells of SCNT embryos were surgically transferred into oviducts of synchronized recipients. The pregnancy status of recipients between days 30 and 35 was determined using ultrasound.

Microsatellite analysis

Parentage analysis was performed on the piglets obtained from somatic cell cloning and the surrogate recipient females to confirm the identity of the donor cells used for nuclear transfer. DNA was extracted from an ear punch or tail clipping obtained from each newborn piglet and the recipients, as well as from the donor cells. Thirteen porcine DNA microsatellite markers (S0005, S00090, S0026, S0155, S0225, SW122, SW24, SW632, SW72, SW787, SW857, SW936, and SW951) were applied to confirm the genetic identity of the cloned piglets to that of the donor cells used for nuclear transfer.

Polymerase chain reaction conditions

A total of 12.5 ng of porcine DNA, 5 pmol of each primer, and 0.1 U of Tag polymerase were included in an 8 [micro]L reaction containing 1x Taq buffer; 1.5 mM Mg[Cl.sub.2]; 30 [micro]M each of dTTP, dGTP, and dCTP; 15 [micro]M dATP, and 0.1 [micro]Ci of [[alpha]-[sup.32]P] dATP. The thermocycler profile was set for 1 min at 92[degrees]C, 28 cycles of 30 s at 94[degrees]C, 1 min at the annealing temperature, 1 min at 72[degrees]C, and a 5-min final extension at 72[degrees]C.

Experimental design

In Experiment 1, different sexes of donor cells (female or male fetal fibroblast cells) at 4 to 10 passages were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence and cell number of reconstructed embryos were examined. In Experiment 2, cloned embryos derived from female or male fetal fibroblasts were surgically transferred to surrogate mothers. When cloned piglets were delivered, we monitored the total duration of the pregnancy, birth weight, and placental weight of the offspring. We performed clinical and pathological examination after delivery and conducted a postmortem analysis of dead piglets. We subjected only recipient pigs and neonatal piglets to neonatal analysis and pathological findings.

Statistical analysis

Differences were analyzed among experiments using one-way analysis of variance after arc-sine transformation of the proportional data. Differences were considered significant at p<0.05.

RESULTS

Experiment 1: Nuclear transfer and development of reconstructed oocytes

The effects of the donor cells on the ability of reconstructed embryos to develop to the blastocyst stage after seven days of in vivo or in vitro culture were evaluated. The blastocyst formation rate of in vitro (11.9% for females vs 11.3% for males) and in vivo (7.2% for females vs 10.6% for males) conditions was not significantly different (p>0.05) between the sexes (Table 1). The mean cell number of in vitro blastocysts (31.4 [+ or -] 8.3 for females vs 33.4 [+ or -] 11.1 for males) was not significantly different (p>0.05) between the sexes. In the two different fetal fibroblast groups, in vivo developmental ability was not significantly different, but the mean cell number of in vivo blastocysts (143.8 [+ or -] 10.5 in females vs 159.2 [+ or -] 14.8 in males) was higher than the in vitro culture of SCNT groups (31.4 [+ or -] 8.3 in females vs 33.4 [+ or -] 11.1 in males). Although the proportions of the reconstructed embryos that developed into in vivo blastocysts were not significantly different between groups using different donor cells, the cell number of in vivo blastocysts was higher than those of in vitro blastocysts (Table 1). In Figure 2, in vivo cultured SCNT blastocysts (A and B) display a higher cell number and more homogeneous cell morphology than in vitro cultured SCNT blastocysts (C and D).

Experiment 2: Surgical embryo transfer and production of cloned piglets

As shown in Table 2, five out of the seven pregnant surrogate gilts that received female reconstructed embryos were allowed to deliver naturally and farrowed on days 115 to 121 of gestation. The birth weight of the 15 female piglets ranged from 0.48 to 1.83 kg. From the remaining two pregnant surrogate gilts that received reconstructed embryos with female donor cells, one pregnant recipient aborted two fetuses on day 46 of gestation and the other aborted during gestation, but the fetuses were inadvertently not recovered. Four of the seven pregnant gilts that received reconstructed embryos with male fibroblast cells produced eighteen male piglets on days 116 to 121 of gestation via vaginal delivery. One pregnant recipient delivered four live male piglets by cesarean section (Table 2), while two pregnant surrogate gilts aborted during gestation, but the fetuses were not recovered. The birth weights of male piglets ranged from 0.45 to 1.50 kg. The average birth weight of the cloned piglets was not significantly different between the sexes (1.36 [+ or -] 0.29 kg in female piglets and 1.22 kg [+ or -] 0.36 in male piglets). The days of gestation length of cloned female and male piglets did not have a significant difference between the sexes (Table 2).

Next, we compared the efficacy of cloning piglets using the female and male fetal fibroblasts as donor cells. The survival rate in the female group (53.3%, 8 out of 15 piglets) was higher than that in the male group (45.5%, 10 out of 22 piglets), but these difference were not statistically significant (Table 3). Characteristics of the cloned piglets are summarized in Table 3. A total of 11 out of 37 cloned piglets (29.7%) died within two weeks following birth. From the 12 cloned female piglets, 1 female piglet died during the first breast-feeding on day 1, and 2 piglets died at days 11 and 14 from first breast-feeding failure of the surrogate. One piglet died from being crushed by the surrogate. A total of 7 out of the 17 cloned male piglets died within a week following birth. Among them, five piglets died between days 1 and 5 from failure to feed, which was related to the cesarean section birth of the surrogate, and two piglets died from diarrhea and crushing by two surrogates, respectively. The total remaining live cloned piglets were 8 female and 10 male, with the survival rate of cloned female and male piglets similar (53.3% and 45.5%, respectively).

Microsatellite analysis using 1 to 13 markers suggested that all 37 piglets were derived from the male or female fetal fibroblast cell line. Parentage analysis was performed on DNA obtained from ear punches of the SCNT piglets and the surrogate recipients to confirm that the piglets were identical to the donor cell line used. Results of the microsatellite marker analysis verified that the donor cell lines were the source of the genetic material used to produce the newborn piglets (Tables 4 and 5).

DISCUSSION

The donor somatic cell karyoplast is one of the important factors affecting the efficiency of somatic cell animal cloning. However, low cloning efficiency has hampered the production of cloned animals. Several studies have compared the effects of different types of donor cells to promote embryo development after SCNT in different species. In mice, an appropriate interaction between cell type and genotype can improve cloning efficiency [22]. In bovines, utilizing cumulus and ear fibroblast cells as donor cells was discovered to have better developmental competence of cloned embryos to the blastocyst stage than embryos reconstructed with uterine or oviductal cells [23]. Cloning with aborted calf-derived donor cells had a higher number of abnormalities than those derived from newborn or fetal cells [18]. Cumulus cells are a more efficient nuclear donor for SCNT than skin fibroblast and granulosa cell lines in buffalos [24]. The type of donor somatic cell is important for the development of cloned embryos; the fetal fibroblasts as a donor cell might be one of the best choices for positive SCNT in pigs [25]. However, comparisons from previous studies reveal that adult cells of any variety are inferior to fetal fibroblasts in terms of reconstructed embryo development. Fetal fibroblasts are highly undifferentiated cells unlike other cells retrieved from adult tissue. The superiority of fetal fibroblasts as shown in several studies might suggest that undifferentiated cells are more amenable to reprogramming after reconstruction than differentiated cells [26,27]. Although a female and male piglet have been produced previously [8], the efficacy of female or male somatic cells as donor nuclei for production of cloned animals has not been well documented. In a previous study, the production efficiencies of cloned miniature pigs using male and female fetal fibroblasts as nuclei donors ranged from 0.64% (2/314) to 0.9% (3/331) via the transfer of reconstructed embryos that had been cultured for 1 to 2 days into miniature and common domestic pigs [20]. In the present study, we investigated the effect of donor cell sex on the efficiency of pig cloning. Our results reveal that 5 recipients delivered 15 female piglets and the other 5 recipients delivered 22 male piglets after the transfer of 11,535 female and male reconstructed embryos into 66 recipients. A total of 18 (8 female and 10 male) piglets survived for greater than 60 days. Although there were no significant differences in pregnancy and delivery rates between the groups, the production rates of cloned piglets derived from the reconstruction of male fibroblast cells were higher than the reconstruction of female fibroblast cells. Considering data from previous studies, the pregnancy rate obtained in the present study (female 17.1% and male 28.0%) after the transfer of eggs matured in vitro was similar to that in studies using in vivo-matured eggs and serial nuclear transfer (29%; [4]) and in vitro-matured oocytes receiving fetal somatic cells (23%; [2]).

In recent years, several studies have reported that the pregnancy rates of SCNT pigs using fetal fibroblasts as nuclei donors ranged from 43% to 100% via transferring of reconstructed embryos cultured for 1 to 2 days into recipients [15,16,20,28]. These studies also discovered that the cell number of female and male in vivo blastocysts was higher than those of in vitro blastocysts, while the average birth weight of the cloned piglets and the day of gestation length of cloned female and male were not significantly different between female and male piglets. In the present study, the survival rates and birth weights of female cloned piglets were higher than those of cloned male piglets. Although the numbers of recipients in other studies were limited, the cloned embryo pregnancies in the present study were lower than those for the pig somatic cell cloning studies with in vivo-matured oocytes [8] and in vitro-matured oocytes [9,29].

Previous studies have reported that SCNT-derived clones are prone to various abnormal phenotypes, including large birth weights [30,31]. Morphological abnormalities of somatic cell cloning have been observed in cloned male piglets [32]. Postnatal death of young and abnormality of male somatic cell cloning was higher than those of female somatic cell cloning in bovines [33]. It has also been reported that a cloned male piglet died from suffocation because of regurgitated ingesta within the respiratory cavities on the day following birth [8]. Some studies have reported that all cloned male piglets appeared quite healthy [2], while other studies have observed a few abnormal phenotypic problems [9,29]. In the present study, the cloned piglets might have a number of physiological defects such as failure of first feeding. Among the cloned piglets that died within two weeks following delivery, male piglets had a higher number (7/17, 41.2%) than did female clones (4/12, 33.3%). Considering data obtained from other studies, the competence of SCNT might be due to the differences of donor cell lines. In particular, the phenotypic abnormality of cloned pigs may be induced by damage during in vitro culture of donor cells. However, whether donor cell types cause multiple-organ failure and sudden early death in cloned males needs to be investigated further. Although we have no explanation for the difference in the cause of death observed between the sexes, further studies are necessary to increase the successful development to term and the survival rate of cloned piglets. In conclusion, the present study indicates that the type of donor cell lines is one of the critical factors for improving the efficiency of SCNT in pigs, while the sex difference might not affect the efficiency of SCNT in pigs.

https://doi.org/10.5713/ajas.16.0591

CONFLICT OF INTEREST

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

ACKNOWLEDGMENTS

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET), 111047-5, http://www.ipet.re.kr/.

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Jae-Gyu Yoo (1,a), Byeong-Woo Kim (2,a), Mi-Rung Park (1), Deug-Nam Kwon (3), Yun-Jung Choi (3), Teak-Soon Shin (2), Byung-Wook Cho (2), Jakyeom Seo (2), Jin-Hoi Kim (3), Seong-Keun Cho (2) *

* Corresponding Author: Seong-Keun Cho

Tel: +82-55-350-5511, Fax: +82-55-350-5519, E-mail: skcho@pusan.ac.kr

(1) Animal Diseases and Biosecurity Team, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea

(2) Department of Animal Science, Life and Industry Convergence Research Institute, Pusan National University, Miryang 627-706, Korea

(3) Department of Animal Biotechnology, KonKuk University, Seoul 143-701, Korea

(a) These authors contributed equally to this work.

Submitted Aug 4, 2016; Revised Sept 12, 2016; Accepted Oct 18, 2016

Caption: Figure 1. Sexing of porcine fetal fibroblast cells by polymerase chain reaction assay. SM, ATGene size marker; 1, fetus 1 (male); 2, fetus 2 (female); and 3, fetus 3 (female); +, positive control (known male gDNA); -, negative control (known female gDNA).

Caption: Figure 2. Representative SCNT blastocysts derived from in vivo cultured (A and B) and in vitro cultured (C and D) SCNT embryos. Scale bar: A and C 100 pm; B and D 200 [micro]m.
Table 1. In vitro and in vivo development of cloned embryos derived
from female and male fetal fibroblast donor cells

Conditions              Number     Number of     Number of
Culture      Sex of     of fused   oocytes       embryos
conditions   donor                 cleaved (%)   developed
             cells (1)                           to
                                                 blastocysts
                                                 (%)

In vitro     Female     211        149 (70.6)    25 (11.9)

             Male       231        158 (68.4)    26 (11.3)

In vivo      Female     1673)      --            12 (7.2)

             Male       1803)      --            19 (10.6)

Conditions              Cell number of
Culture      Sex of     blastocysts (2)
conditions   donor      (range)
             cells (1)

In vitro     Female     31.4 [+ or -]
                        8.3 (17-45)
             Male       33.4 [+ or -]
                        11.1 (19-48)
In vivo      Female     143.8 [+ or -]
                        10.5 (130-1 58)
             Male       159.2 [+ or -]
                        14.8 (147-184)

(1) A total of 4 to 10 passaged porcine fetal fibroblasts. (2)
Mean [+ or -] standard deviation. (3) Number of embryos transferred
into recipient.

Table 2. Production of cloned piglets derived from nuclear transfer
embryos

Sex of     Number of     Number of surrogate recipient  Total number of
donor      embryos       Used   Pregnant   Farrowed     cloned piglets
cells (1)  transferred          (2) (%)     (%)         born (3) (%)
           (range)

Female     6,789         41     7 (17.1)   5 (12.2)   15 (0.22)
           (64-400)
Male       4,746         25     7 (28.0)   5 (20.0)   22 (0.46)
           (100-292)

Sex of     Average birth weight     Days of gestation
donor      of live birth piglets    length (range)
cells 1)   (range)

Female     1.36 [+ or -] 0.29 (a)   118.4 [+ or -] 2.4 (b)
           (0.48-1.83)              (115-121)
Male       1.22 [+ or -] 0.36 (a)   118.0 [+ or -] 2.3 (b)
           (0.45-1.50)              (116-121)

(1) Donor cell line: female, #10 and #1-1; male, #5 and #6 fetal
fibroblast cell.

(2) Female-recipient: two recipients aborted, Male-recipient: two
recipients aborted.

(3) A piglet/embryos transferred eggs: female, 452.6; male, 215.7.

(a,b) Values with different superscripts differ significantly (p
<0.05).

Table 3. Neonatal post-birth characteristics and survival of cloned
piglets produced from female or male fetal fibroblast donor cells

Sex of   Number                 Neonatal conditions      No. of
donor    of        Normal (%)   Abnormal   Stillbirths   piglets
cells    cloned                 (%)        (%)           surviving
         piglets                                         >0 days (%)
         born

Female   15        12 (80.0)    2 (13.3)   1 (6.7)       8 (53.3)
Male     22        17 (77.3)    3 (13.6)   2 (9.1)       10 (45.5)

Table 4. Microsatellite analysis of cloned female piglets derived from
porcine fetal fibroblast cells (1)

Marker   Genotype of recipient         Donor
         RA    RB    RC    RD    RE    cell

PIG_X    218   218   218   218   218   218
         218   218   218   218   218   218
S0005    246   248   246   236   222   238
         252   250   250   250   242   244
S00090   246   250   250   244   250   246
         250   252   254   250   250   250
S0026    104   100   102   100   100   106
         106   106   104   106   106   106
S0155    159   159   163   165   165   163
         163   165   167   167   165   165
S0225    174   174   192   184   174   192
         192   192   192   192   192   192
SW122    130   118   122   118   118   126
         130   122   124   128   130   128
SW24     118   112   118   118   112   118
         124   118   124   118   124   124
SW632    166   168   170   176   168   168
         176   176   176   178   170   180
SW72     113   115   105   115   115   105
         121   117   115   115   121   105
SW787    158   158   160   158   160   156
         158   160   164   160   166   164
SW857    151   147   157   157   157   151
         155   161   161   157   161   155
SW936    102   114   114   102   102   100
         114   116   116   116   108   102
SW951    129   127   129   127   127   127
         129   127   133   135   127   129

Marker   Genotype of litters (female)
         A1    A2    A3    B1    B2    C1

PIG_X    218   218   218   218   218   218
         218   218   218   218   218   218
S0005    238   238   238   238   238   238
         244   244   244   244   244   244
S00090   246   246   246   246   246   246
         250   250   250   250   250   250
S0026    106   106   106   106   106   106
         106   106   106   106   106   106
S0155    163   163   163   163   163   163
         165   165   165   165   165   165
S0225    192   192   192   192   192   192
         192   192   192   192   192   192
SW122    126   126   126   126   126   126
         128   128   128   128   128   128
SW24     118   118   118   118   118   118
         124   124   124   124   124   124
SW632    168   168   168   168   168   168
         180   180   180   180   180   180
SW72     105   105   105   105   105   105
         105   105   105   105   105   105
SW787    156   156   156   156   156   156
         164   164   164   164   164   164
SW857    151   151   151   151   151   151
         155   155   155   155   155   155
SW936    100   100   100   100   100   100
         102   102   102   102   102   102
SW951    127   127   127   127   127   127
         129   129   129   129   129   129

Marker   Genotype of litters (female)
         C2    C3    C4    C5    D1    D2

PIG_X    218   218   218   218   218   218
         218   218   218   218   218   218
S0005    238   238   238   238   238   238
         244   244   244   246   244   244
S00090   246   246   246   246   246   246
         250   250   250   250   250   250
S0026    106   106   106   106   106   106
         106   106   106   106   106   106
S0155    163   163   163   163   163   163
         165   165   165   165   165   165
S0225    192   192   192   192   192   192
         192   192   192   192   192   192
SW122    126   126   126   126   126   126
         128   128   128   128   128   128
SW24     118   118   118   118   118   118
         124   124   124   124   124   124
SW632    168   168   168   168   168   168
         180   180   180   180   180   180
SW72     105   105   105   105   105   105
         105   105   105   105   105   105
SW787    156   156   156   156   156   156
         164   164   164   164   164   164
SW857    151   151   151   151   151   151
         155   155   155   155   155   155
SW936    100   100   100   100   100   100
         102   102   102   102   102   102
SW951    127   127   127   127   127   127
         129   129   129   129   129   129

Marker   Genotype of litters (female)
         D3    E1    E2

PIG_X    218   218   218
         218   218   218
S0005    238   238   238
         244   244   244
S00090   246   246   246
         250   250   250
S0026    106   106   106
         106   106   106
S0155    163   163   163
         165   165   165
S0225    192   192   192
         192   192   192
SW122    126   126   126
         128   128   128
SW24     118   118   118
         124   124   124
SW632    168   168   168
         180   180   180
SW72     105   105   105
         105   105   105
SW787    156   156   156
         164   164   164
SW857    151   151   151
         155   155   155
SW936    100   100   100
         102   102   102
SW951    127   127   127
         129   129   129

(1) Litter A1, 2, and 3 came from recipient RA; Litter B1 and 2 came
from recipient RB; Litter C1, 2, 3, 4, and 5 came from recipient RC;
Litter D1, 2, and 3 came from recipient RD; and Litter E1 and 2 came
from recipient RE.

Table 5. Microsatellite analysis of cloned male piglets derived from
porcine fetal fibroblast cells (1)

Marker   Genotype of recipient         Donor
         RF    RG    RH    RI    RJ    cell

PIG_X    218   218   218   218   218   218
         218   218   218   218   218   218
PIG_Y    --    --    --    --    --    226
         --    --    --    --    --    226
S0005    222   210   242   238   230   234
         242   236   246   246   244   238
S00090   250   248   246   250   250   246
         250   250   250   252   252   248
S0026    100   102   100   100   102   102
         106   104   104   104   104   106
S0155    165   165   165   159   163   163
         165   167   167   165   165   165
S0225    174   174   174   174   188   192
         192   192   192   192   192   192
SW122    118   122   124   128   122   122
         130   122   130   130   122   126
SW24     112   124   104   106   104   118
         124   124   106   124   118   118
SW632    168   168   170   176   176   180
         170   176   178   176   176   180
SW72     115   105   105   105   105   113
         121   115   113   105   115   115
SW787    160   156   158   158   158   156
         166   160   160   158   166   158
SW857    157   147   147   157   147   151
         161   161   155   157   161   155
SW936    102   102   108   102   114   102
         108   108   116   108   116   114
SW951    127   127   127   127   127   127
         127   129   127   127   129   135

Marker   Genotype of litters (male)
         F1    F2    F3    F4    F5    G1    G2    H1    H2    H3

PIG_X    218   218   218   218   218   218   218   218   218   218
         218   218   218   218   218   218   218   218   218   218
PIG_Y    226   226   226   226   226   226   226   226   226   226
         226   226   226   226   226   226   226   226   226   226
S0005    234   234   234   234   234   234   234   234   234   234
         238   238   238   238   238   238   238   238   238   238
S00090   246   246   246   246   246   246   246   246   246   246
         248   248   248   248   248   248   248   248   248   248
S0026    102   102   102   102   102   102   102   102   102   102
         106   106   106   106   106   106   106   106   106   106
S0155    163   163   163   163   163   163   163   163   163   163
         165   165   165   165   165   165   165   165   165   165
S0225    192   192   192   192   192   192   192   192   192   192
         192   192   192   192   192   192   192   192   192   192
SW122    122   122   122   122   122   122   122   122   122   122
         126   126   126   126   126   126   126   126   126   126
SW24     118   118   118   118   118   118   118   118   118   118
         118   118   118   118   118   118   118   118   118   118
SW632    180   180   180   180   180   180   180   180   180   180
         180   180   180   180   180   180   180   180   180   180
SW72     113   113   113   113   113   113   113   113   113   113
         115   115   115   115   115   115   115   115   115   115
SW787    156   156   156   156   156   156   156   156   156   156
         158   158   158   158   158   158   158   158   158   158
SW857    151   151   151   151   151   151   151   151   151   151
         155   155   155   155   155   155   155   155   155   155
SW936    102   102   102   102   102   102   102   102   102   102
         114   114   114   114   114   114   114   114   114   114
SW951    127   127   127   127   127   127   127   127   127   127
         135   135   135   135   135   135   135   135   135   135

Marker   Genotype of litters (male)
         I1    I2    I3    I4    I5    I6    J1    J2    J3    J4

PIG_X    218   218   218   218   218   218   218   218   218   218
         218   218   218   218   218   218   218   218   218   218
PIG_Y    226   226   226   226   226   226   226   226   226   226
         226   226   226   226   226   226   226   226   226   226
S0005    234   234   234   234   234   234   234   234   234   234
         238   238   238   238   238   238   238   238   238   238
S00090   246   246   246   246   246   246   246   246   246   246
         248   248   248   248   248   248   248   248   248   248
S0026    102   102   102   102   102   102   102   102   102   102
         106   106   106   106   106   106   106   106   106   106
S0155    163   163   163   163   163   163   163   163   163   163
         165   165   165   165   167   165   165   165   165   165
S0225    192   192   192   192   192   192   192   192   192   192
         192   192   192   192   192   192   192   192   192   192
SW122    122   122   122   122   122   122   122   122   122   122
         126   126   126   126   126   126   126   126   126   126
SW24     118   118   118   118   118   118   118   118   118   118
         118   118   118   118   118   118   118   118   118   118
SW632    180   180   180   180   180   180   180   180   180   180
         180   180   180   180   180   180   180   180   180   180
SW72     113   113   113   113   113   113   113   113   113   113
         115   115   115   115   115   115   115   115   115   115
SW787    156   156   156   156   156   156   156   156   156   156
         158   158   158   158   158   158   158   158   158   158
SW857    151   151   151   151   151   151   151   151   151   151
         155   155   155   155   155   155   155   155   155   155
SW936    102   102   102   102   102   102   102   102   102   102
         114   114   114   114   114   114   114   114   114   114
SW951    127   127   127   127   127   127   127   127   127   127
         135   135   135   135   135   135   135   135   135   135

Marker   Genotype of litters (male)
         J5

PIG_X    218
         218
PIG_Y    226
         226
S0005    234
         238
S00090   246
         248
S0026    102
         106
S0155    163
         165
S0225    192
         192
SW122    122
         126
SW24     118
         118
SW632    180
         180
SW72     113
         115
SW787    156
         158
SW857    151
         155
SW936    102
         114
SW951    127
         135

(1) Litter F1, 2, 3, 4, and 5 came from recipient RF; Litter G1 and 2
came from recipient RG; Litter H1, 2, and 3 came from recipient RH;
Litter I1, 2, 3, 4, 5, and 6 came from recipient RI; and Litter J1, 2,
3, 4, and 5 came from recipient RJ.
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Author:Yoo, Jae-Gyu; Kim, Byeong-Woo; Park, Mi-Rung; Kwon, Deug-Nam; Choi, Yun-Jung; Shin, Teak-Soon; Cho,
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Date:Apr 1, 2017
Words:6422
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