• 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.

• Korean Journal of Agricultural Science
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• v.48 no.2
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• pp.353-365
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• 2021

Previously, we reported that tissue factor (Tf) was included in the list of differentially expressed genes as an upregulated gene in a rejected porcine heart after xenotransplantation into monkey. In this study, we analyzed that expression of Tf in aortic endothelial cells (pAEC) isolated from alpha 1,3-galactosyltransferase knockout pig in response to allogeneic porcine serum and xenogeneic human serum. The consequence was significant upregulation of Tf expression by responding to human serum compared with porcine serum. To analyze the function of Tf gene as a promoter, we constructed reporter vectors for expression of luciferase linked to 1,246 and 787 base pairs of porcine Tf (pTF1246 and pTF787), and 535 base pairs of human TF (hTF535) sequences including putative promoter regions and AP-1 biding site at the 5' end. The reporter vectors were transfected into pAEC including cytomegalovirus enhancer/chicken β-actin (CAG)-luciferase vector as a control. Luciferase assay showed that all of the promoters were insufficient to express luciferase compared with CAG promoter in basic culture conditions. Notably, pTF1246, pTF787, and hTF535 led to a significant increase of luciferase expression in response to human serum compared with porcine serum while no change of CAG. pTF1246 and pTF787 showed higher expression than hTF535. Taken together, our findings suggest that pTF1246 and pTF787 promoters could mediate target gene expression specifically at xenogeneic stress condition.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.197-201
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• 2011

This study was conducted to investigate the effects of donor cell treatments on the production of transgenic cloned piglets. Ear fibroblast cell obtained from NIH MHC Inbred minipig was used as control. The GalT knock-out/CD45 knock-in (GalT/CD46) transgenic cell lines were established and used as donor cells. The reconstructed GalT/CD46 embryos were surgically transferred into oviduct of naturally cycling surrogate sows (Landrace ${\times}$ Yorkshire) on the second day of standing estrus. Unlike control (1.2 kV/cm, 75.4%), the fusion rate of the GalT/CDl6 donor cells was significantly higher in 1.5 kV/cm, (84.5%) than that of 1.25 kV/cm, (20.2%) (p<0.01). When the number of the transferred embryos were more than 129, the pregnancy and delivery rates were increased to 13/20 (65%) and 5/20 (25%) compared to less then 100 group [1/6 (16.7%) and 0/6 (0%)], respectively. To analyze the effect of donor cell culture condition on pregnancy and delivery rates, the GalT/CD46 donor cells were cultured with DMEM or serum reduced medium. In serum reduced medium group, the pregnancy and delivery rates were improved to 8/12 (66.7%) and 5/12 (41.7%) compared to DMEM group [3/7 (42.9%) and 0/7 (0%)], respectively. In conclusion, it can be postulated that an appropriate fusion condition and culture system is essential factors to increase the efficiency of the production of transgenic cloned piglets.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.161-168
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• 2016

Nucleotide metabolism in endothelium is variable between different species. Recent studies demonstrated that this variability could contribute coagulation dysfunction, even though organs of the alpha 1,3-galactosyltransferase gene knockout pig were transplanted into the primate. CD73 (ecto-5'-nucelotidase) is an enzyme at cell surface catalyzing the hydrolysis of adenosine triphosphate to adenosine, which plays role on a substance for anti-inflammatory and anti-coagulant. Thus, overexpression of CD73 in endothelial cells of the pig is considered as an approach to reduce coagulopathy. In this study, we constructed a human CD73 expression vector under control of porcine Icam2 promoter (pIcam2-hCD73), which is expressed specifically at endothelial cells, and of CMV promoter as a control (CMV-CD73). First, we transfected the CMV-CD73 vector into HEK293 cells, and then confirmed CD73 expression at cell surface by flow cytometry analysis. Next, we transfected the pIcma2-CD73 and CMV-CD73 vectors into primary porcine fibroblasts and endothelial cells. Consequence was that the pIcma2-CD73 vector was expressed only at the porcine endothelial cells, meaning that the pIcam2 promoter lead to endothelial cell-specific expression of CD73 in vitro. Finally, we nucleofected the pIcam2-hCD73 vector into passage 3 fibroblasts, and enforced hygromycin selection of 400mg/ml. We were able to obtain forty three colonies harboring pIcam2-CD73 to provide donor cells for transgenic cloned porcine production.