• 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.

• 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.

• Journal of Chest Surgery
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• v.45 no.6
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• pp.368-379
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• 2012

Background: Bioprostheses for cardiovascular surgery have limitations in their use following as calicification. ${\alpha}$-galactosidase epitope is known as a stimulant of immune response and then shows a progressing calcification. The objective of this study was to evaluate histologic characteristics and mechanical properties of decellularization and treated with ${\alpha}$-galactosidase. Materials and Methods: Bovine pericardial tissues were allocated into three groups: fixation only with glutaraldehyde, decellularization with sodium dodesyl sulfate and decellularization plus treatment with ${\alpha}$-galactosidase. We confirmed immunohistological characteristics and mechanical properties as fatigue test, permeability test, compliance test, tensile strength (strain) test and thermal stability test. Results: Decellularization and elimination of ${\alpha}$-gal were confirmed through immunohistologic findings. Decellularization had decreased mechanical properties compared to fixation only group in permeability (before fatigue test p=0.02, after fatigue test p=0.034), compliance (after fatigue test p=0.041), and tensile strength test (p=0.00). The group of decellularization plus treatment with ${\alpha}$-galactosidase had less desirable mechanical properties than the group of decellularization in concerns of permeability (before fatigue test p=0.043) and strain test (p=0.001). Conclusion: Favorable decellularization and elimination of ${\alpha}$-gal were obtained in this study through immunohistologic findings. However, those treatment including decellularization and elimination of ${\alpha}$-gal implied the decreased mechanical properties in specific ways. We need more study to complete appropriate bioprosthesis with decellularization and elimination of ${\alpha}$-gal including favorable mechanical properties too.

• Reproductive and Developmental Biology
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• v.39 no.3
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• pp.59-67
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• 2015

Galactose-${\alpha}1,3$-galactose (${\alpha}1,3$-Gal) epitope is synthesized at a high concentration on the surface of pig cells by ${\alpha}1,3$-galactosyltransferase gene (GGTA1). The ${\alpha}1,3$-Gal is responsible for hyperacute rejection in pig-to-human xenotransplantation. The generation of transgenic pigs as organ donors for humans is necessary to eliminate the GGTA1 gene that synthesize $Gal{\alpha}$(1,3)Gal. To prevent hyperacute graft rejection in pig-to-human xenotransplantation, previously, we developed ${\alpha}1,3$-galactosyltransferase gene-knock-out somatic cell by homologous recombination. In this study, we established cell lines of ${\alpha}1,3$-GT knock-out expressing hDAF and hHT gene from minipig fibroblasts to apply somatic cell nuclear transfer. The hDAF and hHT mRNA were expressed in the knock-in somatic cells and ${\alpha}1,3$-GT mRNA was suppressed. However, the knock-in somatic cells were increased resistance to human serum-mediated cytolysis.

• Parasites, Hosts and Diseases
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• v.55 no.5
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• pp.505-512
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• 2017

Toxoplasma gondii cathepsin C proteases (TgCPC1, 2, and 3) are important for the growth and survival of T. gondii. In the present study, B-cell and T-cell epitopes of TgCPC1 were predicted using DNAstar and the Immune Epitope Database. A TgCPC1 DNA vaccine was constructed, and its ability to induce protective immune responses against toxoplasmosis in BALB/c mice was evaluated in the presence or absence of the adjuvant ${\alpha}-GalCer$. As results, TgCPC1 DNA vaccine with or without adjuvant ${\alpha}-GalCer$ showed higher levels of IgG and IgG2a in the serum, as well as IL-2 and $IFN-{\gamma}$ in the spleen compared to controls (PBS, pEGFP-C1, and ${\alpha}-GalCer$). Upon challenge infection with tachyzoites of T. gondii (RH), $pCPC1/{\alpha}-GalCer$ immunized mice showed the longest survival among all the groups. Mice vaccinated with DNA vaccine without adjuvant (pCPC1) showed better protective immunity compared to other controls (PBS, pEGFP-C1, and ${\alpha}-GalCer$). These results indicate that a DNA vaccine encoding TgCPC1 is a potential vaccine candidate against toxoplasmosis.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.10
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• pp.1473-1480
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• 2012

The Galactose-${\alpha}1$,3-galactose (${\alpha}1$,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of ${\alpha}1$,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.