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Anukul Taweechaipaisankul,김지은,Jun-Xue Jin,Su Cheong Yeom,Byeong-Chun Lee 대한수의학회 2018 Journal of Veterinary Science Vol.19 No.4
Due to their similarities with humans in anatomy, physiology, and genetics miniature pigs are becoming an attractive model for biomedical research. We aim to establish and evaluate blood type O cells derived from Korean native pig (KNP), a typical miniature pig breed in Korea. Ten cell lines derived from 8 KNP piglets and one adult female KNP (kidney and ear tissues) were established. To confirm the presence of blood type O, genomic DNA, fucosyltransferase (FUT) expression, and immunofluorescence staining were examined. Additionally, fluorescence-activated cell sorting and somatic cell nuclear transfer were performed to investigate the normality of the cell lines and to evaluate their effectiveness in embryo development. We found no significant bands corresponding to specific blood group A, and no increase in FUT expression in cell lines derived from piglets No. 1, No. 4, No. 5, No. 8, and the adult female KNP; moreover, they showed normal levels of expression of a 1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase. There was no significant difference in embryo development between skin and kidney fibroblasts derived from the blood type O KNPs. In conclusion, we successfully established blood type O KNP cell lines, which may serve as a useful model in xenotransplantation research.
Jin, Jun-Xue,Lee, Sanghoon,Taweechaipaisankul, Anukul,Kim, Geon A.,Lee, Byeong Chun S. KARGER AG 2017 CELLULAR PHYSIOLOGY AND BIOCHEMISTRY Vol.41 No.3
<P><B><I>Background/Aims:</I></B> Hypoacetylation caused by aberrant epigenetic nuclear reprogramming results in low efficiency of mammalian somatic cell nuclear transfer (SCNT). Many epigenetic remodeling drugs have been used in attempts to improve <I>in vitro</I> development of porcine SCNT embryos. In this study, we examined the effects of LAQ824, a structurally novel histone acetylase inhibitor, on the nuclear reprogramming and <I>in vitro</I> development of porcine SCNT embryos. <B><I>Methods:</I></B> LAQ824 treatment was supplemented during the culture of SCNT embryos. The reprogramming levels were measured by immunofluorescence and quantified by image J software. Relative expression levels of 18 genes were analyzed by quantitative real-time PCR. <B><I>Results:</I></B> 100 nM LAQ824 treatment of post-activation SCNT embryos for 24 h significantly improved the subsequent blastocyst formation rate. The LAQ824 treatment enhanced histone 3 lysine 9 (H3K9) levels, histone 4 lysine 12 (H4K12) levels, and reduced global DNA methylation levels as well as anti-5-methylcytosine (5-mC) at the pseudo-pronuclear and 2-cell stages. Furthermore, LAQ824 treatment positively regulated the mRNA expression of genes for histone acetylation (<I>HAT1, HDAC1, 2, 3,</I> and <I>6</I>), DNA methylation (<I>DNMT1, 3a</I> and <I>3b</I>), development (<I>Pou5f1, Nanog, Sox2</I>, and <I>GLUT1</I>) and apoptosis (<I>Bax, Bcl2, Caspase 3</I> and <I>Bak</I>) in blastocysts. <B><I>Conclusion:</I></B> Optimum exposure (100 nM for 24 h) to LAQ824 post-activation improved the <I>in vitro</I> development of porcine SCNT embryos by enhancing levels of H3K9 and H4K12, reducing 5-mC, and regulating gene expression.</P>
Stimulatory Effects of Melatonin on Porcine In Vitro Maturation Are Mediated by MT2 Receptor
Lee, Sanghoon,Jin, Jun-Xue,Taweechaipaisankul, Anukul,Kim, Geon-A,Lee, Byeong-Chun MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.6
<P>Melatonin is a multifunctional molecule with numerous biological activities. The fact that melatonin modulates the functions of porcine granulosa cells via the MT2 receptor suggests the possibility of MT2 receptor-mediation for melatonin to promote cumulus expansion of porcine cumulus-oocyte complexes (COCs). Therefore, we investigated the presence of MT2 in porcine COCs, and the effects of melatonin with or without selective MT2 antagonists (luzindole and 4-P-PDOT) on this process; COCs underwent in vitro maturation culturing with six different conditions (control, melatonin, luzindole, 4-P-PDOT, melatonin + luzindole or melatonin + 4-P-PDOT). Cumulus expansion, oocyte nuclear maturation, and subsequent embryo development after parthenogenetic activation (PA) were evaluated. In experiment 1, MT2 was expressed in both oocytes and cumulus cells. In experiment 2, melatonin significantly increased the proportion of complete cumulus expansion (degree 4), which was inhibited by simultaneous addition of either luzindole or 4-P-PDOT. A similar pattern was observed in the expression of genes related to cumulus expansion, apoptosis, and <I>MT2</I>. In experiment 3, no significant difference was observed in immature, degenerate, and MII oocyte rates among the groups. In experiment 4, melatonin significantly increased blastocyst formation rates and total blastocyst cell numbers after PA, but these effects were abolished when either luzindole or 4-P-PDOT was added concomitantly. In conclusion, our results indicate that the MT2 receptor mediated the stimulatory effects of melatonin on porcine cumulus expansion and subsequent embryo development.</P>
Umbilical Hernia and Repair in a Transgenic Male Cloned Pig
Geon A Kim,Jun-Xue Jin,Anukul Taweechaipaisankul,이상훈,Min Jung Kim,이병천 한국임상수의학회 2018 한국임상수의학회지 Vol.35 No.5
We generated a transgenic male cloned pig which was derived from fibroblast of white Yucatan miniaturepig. After 2 weeks of birth, umbilical hernia which was not easily reduced was identified. Considering the usefulnessof cloned pig, surgical treatment for umbilical hernia correction was performed and a cloned pig has been maintainedhealthy. This is the first report and can be useful for the treatments of umbilical hernia of cloned piglets.
Mineralized deposits in the uterus of a pig without pregnancy loss
김지은,Jun-Xue Jin,Anukul Taweechaipaisankul,이상훈,윤병일,조종기,이병천 대한수의학회 2017 Journal of Veterinary Science Vol.18 No.4
Herein, we describe a case of uterine calcification in the uterus of a pig without pregnancy loss. The recipient underwent cloned embryo transfer and Cesarean section for safe delivery of cloned piglets. During the Cesarean section, 4 white, star-like, (2 × 2 × 2) cm, calcified structures were found within the endometrial cavity. Despite dystrophic calcification around the placenta, healthy cloned piglets were produced successfully. To our knowledge, this is the first reported case of dystrophic calcification occurring within the uterus in a pregnant pig.
Generation of CMAHKO/GTKO/shTNFRI-Fc/HO-1 quadruple gene modified pigs
Kim, G. A.,Lee, E. M.,Jin, J. X.,Lee, S.,Taweechaipaisankul, A.,Hwang, J. I.,Alam, Z.,Ahn, C.,Lee, B. C. Chapman & Hall 2017 Transgenic research Vol.26 No.4
<P>As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG(1)-Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc/hHO-1 that will be suitable for xenotransplantation by overcoming hyperacute, acute and anti-inflammatory rejection.</P>
Kim, Geon A.,Jin, Jun-Xue,Lee, Sanghoon,Taweechaipaisankul, Anukul,Oh, Hyun Ju,Hwang, Joing-Ik,Ahn, Curie,Saadeldin, Islam M.,Lee, Byeong Chun Hindawi 2017 BioMed research international Vol.2017 No.-
<P>Soluble human tumor necrosis factor (shTNFRI-Fc) and human heme oxygenase 1 (hHO-1) are key regulators for protection against oxidative and inflammatory injury for xenotransplantation. Somatic cells with more than 10 copy numbers of shTNFRI-Fc and hHO-1 were employed in somatic cell nuclear transfer to generate cloned pigs, thereby resulting in seven cloned piglets. However, produced piglets were all dead within 24 hours after birth. Obviously, postnatal death with liver apoptosis was reported in the higher copy number of shTNFRI-Fc and hHO-1 piglets. In liver, the transcript levels of ferritin heavy chain, light chain, transferrin, and inducible nitric oxide synthase were significantly highly expressed compared to those of lower copy number of shTNFRI-Fc and hHO-1 piglets (<I>P</I> < 0.05). Also, H<SUB>2</SUB>O<SUB>2</SUB> contents were increased, and superoxide dismutase was significantly lower in the higher copy number of shTNFRI-Fc and hHO-1 piglets (<I>P</I> < 0.05). These results indicate that TNFRI-Fc and hHO-1 overexpression may apparently induce free iron in the liver and exert oxidative stress by enhancing reactive oxygen species production and block normal postneonatal liver metabolism. </P>