Until now, problems related to shortage of organ for transplantation have been continuing. Pigs are the most suitable animal for xenotransplantation. Although primates are most similar to humans, they are not suitable because they have low productivity. Pigs are more productive than primates, and their organ size and physiological characteristics are similar to humans, with the exception of primates. In this study, we breeding the transgenic minipigs using natural mating to produce transgenic pigs. And, transgenic pigs has transmission rate that follow mendel’s rule. There are 20% hDAF gene, 20% US11 gene and 50% both hDAF and US11 gene in transgenic offsprings. Furthermore, transgenic pigs followed normal litter size, and piglets also has normal sex ratio. To suppress the immune function, experiments were performed using porcine ear fibroblast that transfected with hDAF and US11gene. In Cytotoxicity experiment against human complement, hDAF gene and double transgenic cell with both hDAF and US11 gene showed effect to reduce cytotoxicity rate in all of human complement condition. US11 gene and double transgenic cell were significantly reduce the cytotoxicity ratio in human NK cell. Besides, hDAF gene transgenic cell also reduce immune response in 10:1 concentration of human NK cell. In conclusion, natural mating was efficient method for breeding transgenic pigs. And, hDAF and US11 genes has effect for reduce cytotoxicity against human NK cell and human complement conditions.

In our previous studies, the cardiac xenotransplantation from an alpha-1,3-galactosyltransferase knockout pig (GT-MCP-MCP) to cynomolgus monkeys showed a mean survival of 38 days. The objective of this study is to genetically upgrade the GT-MCP-MCP pig, to further enhance membrane cofactor protein (MCP) expression and to express an endothelial specific thrombomodulin (TBM). MCP is a complement regulatory protein and TBM is a coagulation inhibitor. As the dicistronic cassette for wild-type-based MCP and TBM concurrent expressions does not show any increase of MCP, we optimized the MCP codon usage (mMCP) and substituted mMCP for MCP. When the mMCP-TBM cassette was transfected to HeLa cells, we were able to find an increased expression of MCP and endothelial cell-specific TBM expression. The cassette was then transfected into ear-skin fibroblasts isolated from one-month-old #23-4 of a GT-MCP-MCP pig, and the cell populations expressing MCP were obtained by MACS cell sorting. We performed a single cell culture of the selected cells, and obtained clones over expressing 90% MCP. The cells of a clone were used as a donor for nuclear transfer and generated GT-MCP/-MCP/mMCP/TBM pig. The transgenic pig was confirmed to be carrying the cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Thus, we believe that the GT-MCP/-MCP/mMCP/TBM transgenic pig would be potential for the prolongation of xenograft survival in the recipients.

This study was performed to compare the healing quality of the allogenic acellular dermal matix (ADM) and xenogenic ADM combined with autologous skin graft. Xenogenic ADM was obtained from two GalT knock-out pigs. Allogenic ADM was obtained from cynomolgus monkeys. ADM was stored with lyophilization. Full-thickness skin wounds were made on the back of two cynomolgus monkeys. In one monkey, wounds were covered by xenogenic ADM combined with autologous skin graft or autologous skin graft only. In another monkey, wounds were covered by allogenic ADM combined with autologous skin graft or autologous skin graft only. Skin healing process was observed during 2 weeks and skin biopsies were performed on 3 months after skin transplantation. We obtained IACUC approval (ORIENT-IACUC-16053) Skin on the xenogenic ADM was necrotized 1 week after skin transplantation. Possibly due to the thickness of ADM, which block the blood supply from the subcutaneous tissue to the autologous skin graft. Skin biopsy revealed that less fibrotic change of the skin on the ADM compared with the skin without ADM. Xenogenic ADM can be used in high degree burn patients who can suffered from contracture after healing since it can reduce fibrotic change.

Alzheimer's disease (AD) has caused by expression of amyloid precursor protein (APP), Tau and presenilin (PS) as known as plaques and tangle accumulation. AD transgenic porcine model is necessary for preclinical testing of therapeutic agent because of similar metabolic system between porcine and human. The objective of study was to generate AD transgenic pig by somatic cell nuclear transfer (SCNT) with multi-cistronic vector system. AD multi-cistronic vector was 6 well-known mutation on 3 AD related genes, hAPP (K670N/M671L, I716V, V717I), hTau (P301L) and hPS1 (M146V, L286P). Establishment of AD transgenic cell lines was used from Jeju black pig ear fibroblast cells (JB-PEFAD) with the AD multi-cistronic vector. The JB-PEFAD cell was confirmed on mRNA expression, protein synthesis of hAPP, hTau and hPS1 and identification of integration and karyotype. Although fusion rate was no difference in SCNT with JB-PEF AD (SCNTAD) embryos, cleavage and blastocyst formation rates were slightly lower than in SCNT with non-transgenic JB-PEF (SCNTnon-TG). Individual SCNTAD blastocysts were detected hAPP, hTau and hPS1 genomic integration which showed 93.2% (n=30) efficiency in genomic DNA (gDNA) level. It will give us a possibility to develop porcine animal model for AD study in the future.

Xenotransplantation of pig islet regarded as a good alternative to allotransplantation. However, cellular death mediated by hypoxia-reoxygenation injury after transplantation disturb success of this technique. In the present study, we produce transgenic pig expressing human heme oxygenase 1 (HO1) genes to overcome cellular death for improving efficiency of islet xenotransplantation. Particularly, Korean miniature pig breed, Micro-Pig, was used in the present study. Somatic cell nuclear transfer (SCNT) technique was used to produce the HO1 transgenic pig. Six alive transgenic piglets were produced and all the transgenic pigs were founded to have transgene in their genomic DNA and the gene was expressed in all tested organs. Also, in vitro cultured fibroblasts derived from the HO1 transgenic pig showed low reactive oxygen species level, improved cell viability and reduced apoptosis level

Understanding the behavior of transgenes introduced into oocyte or embryos is essential for evaluating the methodologies for transgenic animal production. To date, many studies have reported the production of transgenic pig embryos with, however, low efficiency in environment of blastocyst production. The aim of present study was to determine the expression and duration of transgene transferred by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of GFP and then for the transmission of the transgene. Briefly, fresh spermatozoa were bound to exogenous DNA after treatment by Triton X-100 and Lipofectin. When ICSI-MGT was performed using sperm heads with tails removed, the yield of blastocyst (25.3%), treated with Lipofectin (18.8%) and Triton X-100 (19.2%) were observed. Treatments of Lipofectin or Triton X-100 did not further improve the rates of blastocysts. Moreover, the apoptosis rates of embryos were obtained from the control and LIpofectin groups (8.7%, 9.7%, respectively), but were significantly higher in the Triton X-100 group (13.0%). Our results demonstrated that ICSI-MGT caused minimal damage to oocytes that could develop to full term. Moreover, the embryos derived by ICSI-MGT have shown prolonged exogenous DNA expression during preimplantation stage in vivo. However, more efforts will be required to improve the procedures of both sperm treatments cause of high frequency of mosaicisms.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. Therefore, we applied CRISPR/Cas9 system to generate FoxN1-targeted pig parthenogenetic embryos. Using single guided RNA targeted to pig FoxN1 genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. In results, regardless of the concentrations of vector, the cleavage rate were significantly (p<0.05) decreased (4 ng/μl, 51.24%; 8 ng/μl, 40.88%; and 16 ng/μl; 45.22%) compared to no injection group (70.44%). The blastocyst formation rates were also decreased in vector injected 3 groups (4 ng/μl, 7.96%; 8 ng/μl, 6.4%; and 16 ng/μl; 9.04%) compared to no injection group (29.07%). In addition, the blastocyst formation rates between sham injected group (13.51%) and no injection group (29.07%) also showed significant difference (p<0.05). The mutation rates were comparable between groups (4 ng/μl, 18.4%; 8 ng/μl, 12.5%; and 16 ng/μl; 20.0%). The sequencing analysis showed that blastocysts derived from each group were successfully mutated in FoxN1 loci regardless of the vector concentrations. However, the deletion patterns were higher than the patterns of point mutation and insertion regardless of the vector concentrations. In conclusion, we described that cytoplasmic microinjection of FoxN1-targeted CRISPR/Cas9 vector could efficiently generate transgenic pig parthenogenetic embryos in one-step.

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 α1,3-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.

Hyperacture rejection (HAR) of pig organs, upon xenotransplantation into primates, could partly be overcome by knocking out the alpha-Gal gene. However, xenotransplanted organs may still undergo immunological acture rejection (AR) or acute vascular rejection (AVR). Among several genes involved in AR and AVR, the hCD47 evades the monocyte/ macrophage mediated phagocytosis by identifying the self/non-self signal (CD47-SIRPa) whereas hTFPI participates in the regulation of coagulation pathway by acting upstream of the thrombin. In this study, we investigated hCD47 and hTFPI as two possible candidates for avoiding AR and AVR, respectively upon pig-to-human xenotransplantation. A co-expression vector for hCD47 and hTFPI was constructed using 2A peptides system (F2A) and transfected into the porcine kidney cell line (PK-15). The transfected cells stably expressed both hCD47 and hTFPI mRNA and proteins. Co-culture of non-transfected, hCD47-transfected, hTFPI-transfected or hCD47+hTPFI-transfected PK15 cells with natural killer (NK) cells, monocytes and macrophages confirmed the cytotoxic effect of hCD47 and revealed a synergistic effect of hCD47 and hTFPI co-transfection. There was an attractive survivability of 25～30% on each type of innate immune cell, NK cell and macrophage. These results suggest that transgenic pigs, genetically modified for hCD47 and hTFPI may be useful for overcoming xenograft rejection. Furthermore, cotransfection with hTFPI may enhance the cytotoxic effect of hCD47, possibly by assisting the hCD47-SIRPa binding by an unknown mechanism.

The overexpression of Phosphoprotein Enriched in Astrocytes (PEA15) gene is commonly found in human diabetic patients. The overexpression of this gene in skeletal muscle and fat tissues have been reported to cause insulin resistance, thereby impairing insulin stimulated glucose uptake. We introduced a gene of mouse PEA15 (mPEA15) and enhanced green fluorescent protein (EGFP) into fertilized one cell pig zygotes using microinjection, and produced a piglet that showed overexpression of mPEA15 in the muscle tissues and expression of EGFP in the ear tissues and hooves. RT-PCR RFLP, southern blot and FISH analysis showed that the tissues carried the transgene. Real-time RT-PCR and western blots demonstrated that PEA15 gene was overexpressed in the various tissues and muscle tissues, respectively. These facts suggest that expression vector system is normally expressed in the trnasgenic (TG) pigs. To use as animal diseases model for type 2 diabetes, further study is necessary to confirm whether diabetes occur in these TG pigs, especially insulin resistance.

Our previous study showed that transgenic (TG) pigs harboring human EPO (hEPO) gene have been shown to have reproductive disorders, including low pregnancy rates, irregular estrus cycle and low little size. To investigate these reasons, we assessed estrus behavior (standing response) and plasma 17B-estradiol (E2) level, which partly reflect reproductive function, during the estrus cycles after synchronization and superovulation by hormone treatments. Then, we analysed blood composition and expression of hEPO gene in TG pigs. Pigs were injected with PG600. After 10 days, pigs were fed with Regumate porcine for 6 days. Blood samples were collected from jugular vein. Analysis of blood composition and E2 level were measured by Hemavet 950 and E2 ELISA kit, respectively. And, the expression of hEPO gene in reproductive organs was quantitated by real-time RT-PCR. The percentage of estrus behavior in TG was significantly decreased. Hematocrit (HCT), hemoglobin (Hb) concentration and red blood cell (RBC) number were significantly higher in TG than wild type (WT). On the other hand, high expression of hEPO gene in TG was observed in the mammary gland as well as in the uterus. Moreover, plasma E2 level was significantly higher in TG than WT. These results suggest that nonspecific expression of hEPO gene in the other organs of TG may affect blood composition and plasma E2 level, thereby causing reproductive disorders.