• Journal of Embryo Transfer
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• v.32 no.3
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• pp.165-170
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• 2017

Pigs have been extensively used as mediators of xenotransplantation research. Specifically, the Massachusetts General Hospital (MGH) miniature pig was developed to fix major histocompatibility antigens for use in xenotransplantation studies. We generated transgenic pigs for xenotransplantation using MGH pigs. However, it has not been studied yet whether these pigs show similarity of reproductive physiological characteristics to wild types of MGH miniature pig. In this study we analyzed the estrous cycles and pregnancy characteristics of wild type (WT) and transgenic MGH miniature pigs, which were ${\alpha}1,3$-galactosyltransferase (GalT) heterozygous and homozygous knock-out, and membrane cofactor protein (MCP) inserted in its locus, $GalT^{-MCP/+}$ and $GalT^{-MCP/-MCP}$ pigs. Estrous cycles of WT, $GalT^{-MCP/+}$ and $GalT^{-MCP/-MCP}$ pigs were $20.9{\pm}0.74$, $20.1{\pm}1.26$, and $17.3{\pm}0.87days$, respectively, and periods of estrous were $3.2{\pm}0.10$, $3.1{\pm}0.12$, and $3.1{\pm}0.11days$. The periods of gestation of WT, $GalT^{-MCP/+}$ and $GalT^{-MCP/-MCP}$ pigs were $114.2{\pm}0.37$, $113.3{\pm}0.67$, and $115.4{\pm}0.51days$, respectively. Litter sizes of WT, $GalT^{-MCP/+}$ and $GalT^{-MCP/-MCP}$ pigs were $4.8{\pm}0.35$, $4.8{\pm}1.11$ and $3.0{\pm}0.32$ respectively. There were no significant differences on estrous cycle, periods of estrous and gestation, and litter size among WT, $GalT^{-MCP/+}$ and $GalT^{-MCP/-MCP}$ pigs, meaning that GalT knock-out and additional expression MCP of the MGH miniature pig did not effect on reproduction traits. These results provide relevant information to establish breeding system for MGH transgenic pig, and for propagation of $GalT^{-MCP/-MCP}$ pig to supply for xenotransplantation research.

• Reproductive and Developmental Biology
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• v.39 no.4
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• pp.127-132
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• 2015

To overcome the hyperacute immune rejection during pig-to-non-human primates xenotranasplantation, we have produced and bred ${\alpha}$-1,3-galactosyltransferase knock-out ($GalT^{-/-}$) pigs. In this study, the somatic cells and tissues from the $GalT^{-/-}$ pigs were characterized by an analysis of the expression of Gal${\alpha}$-1,3-Gal (${\alpha}-Gal$) epitope. Briefly, ear fibroblast cell lines of 19 homozygous $GalT^{-/-}$ pigs were established and cryopreserved. The expression of ${\alpha}-Gal$ epitope in the cells was measured by fluorescence activated cell sorter (FACS) analysis using BS-I-B4 lectin. Also, the homozygous ($GalT^{-/-}$) cells and tissues samples were immunostained with BS-I-B4 lectin for analysis of ${\alpha}-Gal$ epitope expression. The results showed that the expression of ${\alpha}-Gal$ epitope in $GalT^{-/-}$ cells (0.2 %) were significantly (p<0.05) down-regulated to the range of cynomolgus monkey fibroblast (0.2 %) cells compared to heterozygous ($GalT^{-/+}$) (9.3 %) and wild type ($GalT^{+/+}$) (93.7 %) fibroblast cells. In the immunostaining results, while the expression of ${\alpha}-Gal$ epitope was detected a partly in $GalT^{-/+}$ cells and mostly in $GalT^{+/+}$ cells, it was almost not detected in the $GalT^{-/-}$ cells. Also, immunostaining results from various tissues of the $GalT^{-/-}$ pig showed that the expression of ${\alpha}-Gal$ epitope was not detectable, whereas various tissues from $GalT^{+/+}$ pig showed a strong expression of ${\alpha}-Gal$ epitope. Our results demonstrated that ${\alpha}-Gal$ epitope expressions from $GalT^{-/-}$ pigs were successfully knocked out to prevent hyperacute immune rejection for further study of xenotransplantation.

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.333-337
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• 2014

To compensate for the critical shortage of human organs for allotransplantation, xenotransplantation studies using genetically modified pigs are being performed in Korea. Two types of pigs that are used are ${\alpha}1,3$-galactosyltransferase gene knockout (GalT KO) pigs and GalT KO+hCD46 (human complement regulatory protein) pigs. The present study measured the gestation time, birth weight, daily growth rate, and heart weight of both kinds of transgenic minipigs. The gestation period for both types of pigs was 117~119 days. There was no difference in the body weight of GalT KO (-/+) and GalT KO (-/-) piglets, but GalT KO+hCD46 ($-^{hCD46+}/+$) pigs were significantly heavier at birth than were GalT KO+hCD46 ($-^{hCD46+}/-^{hCD46+}$) pigs. During the first 10 weeks of life, the daily weight gain of GalT KO+hCD46 ($-^{hCD46+}/-^{CD46+}$) piglets, which are considered the optimal type for xenotransplantation, was 0.19 kg. The weight of hearts from GalT KO piglets up to two months of age was affected more by body weight than by age. Transgenic pigs showed no differences in gestation period or reproductive ability compared with normal pigs. These results comprise basic data that may be used in xenotransplantation studies and transgenic animal production in Korea.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.227-233
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• 2017

Pig has been known to be one of the most feasible animals as a bioreactor to produce pharmaceuticals in milk and as a mediator in xenotransplantation research. Previously, we generated transgenic pigs for both purposes, which were expressing Factor 8, vWF, hTPA, and hEPO in milk, along with expression of MCP at GalT gene locus ($GalT^{-MCP/-MCP}$) as well as expressing MCP at GalT gene loci with CD73 expression ($GalT^{-MCP/+}/CD73$). In this study, we performed comparative analyses of sperm parameters between wild type male (WT) pig and those transgenic males to examine the effects of transgenes integrated into the pigs on motility, morphology, viability, and acrosome integrity of the spermatozoa. Our results showed that the rates of actively motile spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 85.0%, 83.3%, 82.5%, 83.3%, 82.5%, 77.5%, and 78.7%, respectively. Whereas, the rates of morphologically normal spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 90.0%, 80.0%, 80.0%, 83.3%, 85.0%, 91.8%, and 80.8%, respectively. In addition, the viability in spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 93.9%, 82.4%, 89.9%, 83.9%, 87.4%, 92.8%, and 83.6%, respectively. The rates of spermatozoa with normal acrosome integrity in WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 98.1%, 98.6%, 98.6%, 98.7%, 98.1%, 99.5%, and 95.1%, respectively. There were no significant differences in motility, morphology, viability, and acrosome integrity of the spermatozoa among WT, Factor 8, vWF, hTPA, and hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs. These mean that neither random integration nor targeted integration of the transgene into chromosome of pig effect on characteristics of spermatozoa. Ultimately, the transgenic male pigs subjected in this study could apply to propagate their progenies for production of human therapeutic proteins and advancing the xenotransplantation research.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.511-518
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• 2011

To avoid hyperacute rejection of xenografts, ${\alpha}1,3$-galactosyltransferase knock-out (GalT KO) pigs have been produced. In this study, we examined whether Sia-containing glycoconjugates are important as an immunogenic non-Gal epitope in the pig liver with disruption of ${\alpha}1,3$-galactosyltransferase gene. The target cells were then used as donor cells for somatic cell nuclear transfer (scNT). A total of 1,800 scNT embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. Real-time RT-PCR and glycosyltransferase activity showed that ${\alpha}2,3$-sialyltransferase (${\alpha}2,3ST$) and ${\alpha}2,6$-sialyltransferase (${\alpha}2,6ST$) in the heterozygote GalT KO liver have higher expression levels and activities compared to controls, respectively. According to lectin blotting, sialic acidcontaining glycoconjugate epitopes were also increased due to the decreasing of ${\alpha}$-Gal in heterozygote GalT KO liver, whereas GalNAc-containing glycoconjugate epitopes were decreased in heterozygote GalT KO liver compare to the control. Furthermore, the heterozygote GalT KO liver showed a higher Neu5Gc content than control. Taken together, these finding suggested that the deficiency of GalT gene in pigs resulted in increased production of Neu5Gc-bounded epitopes (H-D antigen) due to increase of ${\alpha}2,6$-sialyltransferase. Thus, this finding suggested that the deletion of CMAH gene to the GalT KO background is expected to further prolong xenograft survival.

• Molecules and Cells
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• v.38 no.1
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• pp.65-74
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• 2015

Carbohydrate antigens expressed on pig cells are considered to be major barriers in pig-to-human xenotransplantation. Even after ${\alpha}1,3$-galactosyltransferase gene knock-out (GalT-KO) pigs are generated, potential non-Gal antigens are still existed. However, to the best of our knowledge there is no extensive study analyzing N-glycans expressed on the GalT-KO pig tissues or cells. Here, we identified and quantified totally 47 N-glycans from wild-type (WT) and GalT-KO pig fibroblasts using mass spectrometry. First, our results confirmed the absence of galactose-alpha-1,3-galactose (${\alpha}$-Gal) residue in the GalT-KO pig cells. Interestingly, we showed that the level of overall fucosylated N-glycans from GalT-KO pig fibroblasts is much higher than from WT pig fibroblasts. Moreover, the relative quantity of the N-glycolylneuraminic acid (NeuGc) antigen is slightly higher in the GalT-KO pigs. Thus, this study will contribute to a better understanding of cellular glycan alterations on GalT-KO pigs for successful xenotransplantation.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.9-14
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• 2012

This study was conducted to analyze the transgenic efficiency and sex ratio in ${\alpha}$-1,3-galactosyltransferase (GalT) knock-out (KO) transgenic pigs according to generation. GalT KO piglets were produced by artificial insemination or natural mating. The transgenic confirmation of GalT KO was evaluated by PCR amplification using specific primers. After electrophoresis, three types of bands were detected such as 2.3 kb single band (Wild), 2.3 and 3.6kb double bands (GalT KO -/+; heterozygote), and 3.6kb single band (GalT KO -/-; homozygote). Transgenic efficiency in F1 generation was 64.5% (23/35) of GalT KO (-/+). In F2 generation, GalT KO transgenic efficiency was 36.4% (21/57, Wild), 47.5% (28/57, GalT KO -/+), and 16.1% (8/57, GalT KO -/-), respectively. Interestingly, no homozygote piglets were born in 6 deliveries among total 11 deliveries, although they were pregnant between male (M) and female (F) $F_1$ heterozygote. In the 5 litters including at least one GalT KO -/- piglet, the transgenic efficiency was 13.3% (2/24, Wild), 51.3% (14/24, GalT KO -/+), and 35.3% (8/24, GalT KO -/-), respectively. The sex ratio of M and F was 40:60 in $F_1$ and 49:51 in $F_2$ generation, respectively. Based on these results, GalT KO transgenic pigs have had a reproductive ability with a normal range of transgenic efficiency and sex ratio.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.73-79
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• 2017

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous ${\alpha}1,3$-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig ($GalT^{-MCP/+}$), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig ($GalT^{-MCP/-MCP}$) by crossbreeding $GalT^{-MCP/+}$ pigs. Two female founders gave birth to six of $GalT^{-MCP/-MCP}$, and seven $GalT^{-MCP/+}$ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the $GalT^{-MCP/-MCP}$ pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the $GalT^{-MCP/-MCP}$ pig is a useful candidate to apply xenotransplantation study.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.249-255
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• 2015

Diabetes mellitus, the most common metabolic disorder, is divided into two types: type 1 and type 2. The essential treatment of type 1 diabetes, caused by immune-mediated destruction of ${\beta}-cells$, is transplantation of the pancreas; however, this treatment is limited by issues such as the lack of donors for islet transplantation and immune rejection. As an alternative approach, stem cell therapy has been used as a new tool. The present study revealed that bone marrowderived mesenchymal stromal cells (BM-MSCs) could be transdifferentiated into pancreatic cells by the insertion of a key gene for embryonic development of the pancreas, the pancreatic and duodenal homeobox factor 1 (PDX1). To avoid immune rejection associated with xenotransplantation and to develop a new cell-based treatment, BM-MSCs from ${\alpha}$-1,3-galactosyltransferase knockout (GalT KO) pigs were used as the source of the cells. Transfection of the EGFP-hPDX1 gene into GalT KO pig-derived BM-MSCs was performed by electroporation. Cells were evaluated for hPDX1 expression by immunofluorescence and RT-PCR. Transdifferentiation into pancreatic cells was confirmed by morphological transformation, immunofluorescence, and endogenous pPDX1 gene expression. At 3~4 weeks after transduction, cell morphology changed from spindle-like shape to round shape, similar to that observed in cuboidal epithelium expressing EGFP. Results of RT-PCR confirmed the expression of both exogenous hPDX1 and endogenous pPDX1. Therefore, GalT KO pig-derived BM-MSCs transdifferentiated into pancreatic cells by transfection of hPDX1. The present results are indicative of the therapeutic potential of PDX1-expressing GalT KO pig-derived BM-MSCs in ${\beta}-cell$ replacement. This potential needs to be explored further by using in vivo studies to confirm these findings.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.87-94
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• 2017

Tissue engineering (TE) has been developed to create functional organs and tissue by combining 3D matrix and cells in vitro. Vascularization and angiogenesis are utmost important for supply of nutrients and oxygen in tissue engineered organs. The present study was performed to isolate and characterize primary endothelial cells (EC) from aorta of alpha 1, 3-enzyme galactosyltransferase knock out (GalT KO) pig, to minimize immune rejection and analyze body immune system for future xenotransplantation studies. Isolation of primary EC from aorta were performed by incubation with dispase for 8-10 min at $37^{\circ}C$. Primary EC were cultured in EC growth medium on different extra cellular matrix (ECM), either collagen or gelation. Primary EC exhibits morphological characteristics and showed positive expressions of EC specific marker proteins i.e. PECAM1, KDR and VWF despite of their ECM surface; however, on collagen based surface they showed increase in mRNA level analyzed by qPCR. Primary EC cultured on collagen were sorted by flow cytometer using KDR marker and cultured as KDR positive cells and KDR negative cells, respectively. KDR positive cells showed dramatically increased in PECAM1 and VWF level as compared to KDR negative cells. Based on the above results, primary EC derived from GalT KO are successfully isolated and survived continuously in culture without becoming overgrown by fibroblast. Therefore, they can be utilize for xeno organ transfer, tissue engineering, and immune rejection study in future.