• Journal of Embryo Transfer
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• v.30 no.4
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• pp.345-350
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• 2015

In the last 10 years, porcine somatic cell nuclear transfer to generate transgenic pig has been performed tremendous development with introduction and knockout of many genes. However, efficiency of porcine somatic cell nuclear transfer is still low and embryo transfer (ET) is one of important step for production efficiency. In porcine ET for production of transgenic cloned pig, we can consider many of points to increase production rates. In respect of seasonality and weather, porcine ET usually is not performed in summer and winter. Cloned transgenic embryos must be transferred into reproductive tracts of recipients where embryos are located after natural fertilization with similar estrous cycle. If cloned embryos with 2~4 cell stage are transferred, they must be transferred into oviducts in periovulatory stage. Number and deposition sites of transferred cloned embryos are important. And we must compare the methods of ET between surgical and non-surgical ones in respect of production efficiency. Sow recipients after natural estrus is most preferred recipients however its cost is must be considered. Here we will review many of current studies about porcine embryo transfer to increase production efficiency of transgenic pigs and strategies for further studies.

• 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.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.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.3
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• pp.324-329
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• 2014

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells ($RFP^+/eGFP^+$) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

• 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.