Pigs have been extensively used as mediators of xenotransplantation research. Specifically, the Massachusetts General Hospital (MGH) miniature pig was developed to fix major histocompatibility antigens for use in xenotransplantation studies. We generated transgenic pigs for xenotransplantation using MGH pigs. However, it has not been studied yet whether these pigs show similarity of reproductive physiological characteristics to wild types of MGH miniature pig. In this study we analyzed the estrous cycles and pregnancy characteristics of wild type (WT) and transgenic MGH miniature pigs, which were α1,3-galactosyltransferase (GalT) heterozygous and homozygous knock-out, and membrane cofactor protein (MCP) inserted in its locus, GalT-MCP/+ and GalT-MCP/-MCP pigs. Estrous cycles of WT, GalT-MCP/+ and GalT-MCP/-MCP pigs were 20.9±0.74, 20.1±1.26, and 17.3±0.87 days, respectively, and periods of estrous were 3.2±0.10, 3.1±0.12, and 3.1±0.11 days. The periods of gestation of WT, GalT-MCP/+ and GalT-MCP/-MCP pigs were 114.2±0.37, 113.3±0.67, and 115.4±0.51 days, respectively. Litter sizes of WT, GalT-MCP/+ and GalT-MCP/-MCP pigs were 4.8±0.35, 4.8±1.11 and 3.0±0.32 respectively. There were no significant differences on estrous cycle, periods of estrous and gestation, and litter size among WT, GalT-MCP/+ and GalT-MCP/-MCP pigs, meaning that GalT knock-out and additional expression MCP of the MGH miniature pig did not effect on reproduction traits. These results provide relevant information to establish breeding system for MGH transgenic pig, and for propagation of GalT-MCP/-MCP pig to supply for xenotransplantation research.

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous α 1,3-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig (GalT-MCP/+), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig (GalT-MCP/-MCP) by crossbreeding GalT-MCP/+ pigs. Two female founders gave birth to six of GalT-MCP/-MCP, and seven GalT-MCP/+ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the GalT-MCP/-MCP pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the GalT-MCP/-MCP pig is a useful candidate to apply xenotransplantation study.

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.

The current study was conducted to evaluate the biocompatibility of α-1,3 galactosyltransferase knockout pig bone graft in a rat calvarial defect model. Porcine cancellous bones were harvested from general and alpha-gal KO pigs and washed with 70% ethanol solution and normal saline. Bone pieces of the alpha-gal KO pig underwent a chemical treatment process to delipidize and deproteinize the bone. Bone graft particles were freeze-dried and stored at −70°C until use. Each bone graft was implanted into the rat calvarial defect in a fresh general pig, fresh transgenic pig, and chemical-treated pig bone group. There was no systemic adverse effect on hematology or necropsy findings in all groups at 1 week and 4 weeks. In the microcomputed tomography analysis, bone volume increased significantly in the chemical-treated transgenic pig bone group, whereas bone mineral density decreased significantly in the fresh general pig bone group compared with other groups. Histological evaluation showed cellular infiltration located at the margin of the bone graft particles, especially in the fresh general pig bone group. These results indicate that fresh general pig bone can elicit a greater local inflammatory response than fresh transgenic pig bone. Further, chemical-treated transgenic pig bone graft was less immunogenic than fresh bone graft. In conclusion, transgenic pig bone is a more biocompatible graft material. In addition, chemical treatment can reduce bone graft immunogenicity by delipidizing and deproteinizing bone.

Transgenic pigs are promising donor organisms for xenotransplantation as they share many anatomical and physiological characteristics with humans. Recently, a step has been moved closer to xenotransplantation by producing genetically modified pigs that has no α-1,3-Gal epitope, the major xenoantigens triggering HAR of pig to primate xenografts. Further genetic modifications such as expression of human complementary regulatory proteins, CD39, endothelial protein C receptor, heme-oxygenase 1, thrombomodulin, tissue factor pathway inhibitoras well as modulators of the HLA-E/β-2-microglobulin, and CTLA-4Ig are due to address for further rejection mechanisms and incompatibilities between porcine and primate blood coagulation systems. Although the pig is the favored species for use as a xenograft donor, a detailed description of the transgenic pig development and surgical technique is lacking which seems mandatory to address for broader understanding of this issue.

장기이식분야는 효과적인 면역억제제의 개발과 더불어 비약적으로 발전하여 현재는 다양 한 장기이식이 활발히 시행되고 있다. 그러나 면역억제제의 장기 복용으로 인하여 감염은 물론 장기적으로 암이나 심혈관계 질환 등의 합병증은 여전히 문제가 되고 있다. 이를 극복 하기 위해서 다양한 시도들이 이루어지고 있다. 첫째는 고형 장기이식과 동시에 골수이식을 시행하여 면역관용을 유도하고 면역억제제 복용을 중단함으로써 약제에 의한 장기 부작용을 차단하려는 시도이다. 둘째는 현재도 여 러 장기이식들에서 면역억제제를 끊고도 이식 장기의 기능이 유지되는 임상적으로 면역관용 상태인 환자들이 있다. 면역관용 상태를 감시할 수 있는 검사법을 개발한다면 향후 환자들 에서 이식 후 주기적으로 면역관용 검사를 시행하고 관용 상태로 나타날 경우, 면역억제제 를 끊는 시도를 할 수 있고 면역관용 검사에서 관용으로 갈 확률이 낮을 경우 지속적으로 면역억제제를 사용하는 환자 개인별 맞춤형 면역억제 치료를 할 수 있을 것으로 기대한다. 한편 이런 기술들이 발달하더라도 고령화와 만성병의 증가로 공여 장기의 부족은 여전히 중요한 문제가 될 전망이기 때문에, 이종장기를 이용하여 장기이식의 수급 불균형을 해소 하 고자 하는 시도가 있다. 하지만, 이종이식의 경우 동종 이식 시 보다 격렬한 거부반응이 발 생하는 것으로 알려져 있으며, 돼지에 다량 존재하는 알파갈에 대한 인간의 자연항체 반응 에 의한 초급성 거부반응이 발생하여 이식된 장기가 수분 또는 수시간 내 급속히 파괴된다. 현재 초급성거부반응은 알파갈 적중 돼지의 개발과 인간의 혈청보체 조절인자를 발현하는 형질전환돼지의 개발로 어느 정도 극복이 되었지만, 초급성거부반응 극복 이후에도 여전히 급성혈관성 거부반응이 발생하며, 혈관내피세포가 2차적으로 활성화되어 선천성 면역세포 의 침윤과 함께 혈액응고 현상이 나타난다. 따라서 요즈음은 초급성거부반응 이후의 다양한 급성혈관성 거부반응을 조절하기 위한 다중 형질전환 무균돼지를 생산하는 기술이 개발되었다. 알파갈이 결손된 돼지의 장기를 영 장류에 이식한 경우에 혈관 내피세포가 활성화되어 섬유소가 침착되며, 선천성 면역반응계 세포의 침윤이 일어나는 것이 보고된 바 있다. 이러한 거부반응을 극복하기 위해서 선천성 면역반응 조절과 함께 항응고 유전자를 과발현 시키거나, 섬유소 분해를 촉진하는 유전자 를 도입한 형질전환돼지의 개발이 시도되고 있으며 우리나라에서도 이 분야 연구가 진행 중이 다. 장기이식에 비해 췌도 이식은 혈관을 연결하는 과정이 없기 때문에 이종이식거부반응의 강도가 약하고 시술의 위험이 적어서 전 세계적으로 이종췌도이식에 대한 연구가 활발히 진행되고 있다. 특히, 2009년 데이비드 쿠퍼 그룹에서는 혈청보체 조절인자인 hCD46를 발 현하는 형질전환돼지의 췌도를 영장류에 이식하였을 때 최장 1년까지 인슐린 비의존성을 보인바 있으며 벨기에 듀프렌은 돼지의 췌도를 생체 친화적 소재로 캡슐레이션하여 영장류 에 이식하였고 1년 가까운 생존율을 보고하였다. 이와 같은 이종장기이식은 장기이식 분야 의 중요한 미래 기술로서 장기 기증에 제한적인 문화배경을 가지고 있으며, 고령화 사회에 급속히 들어서는 우리나라에 필수적인 분야로 생각되며, 췌도, 파킨슨병, 각막 이식 등으로 부터 시작하여 고형장기로 그 시도가 확대될 것으로 전망된다. 한편, 다른 방향으로 줄기세포를 이용하여 분화시킴으로써 세포이식에 이용하려는 노력도 계속되고 있다. 최근 역분화 줄기세포 유도 기술이 발달됨에 따라 향후 세포이식 분야와의 접목이 기대되고 있다.

This study was conducted to analyze the transgenic efficiency and sex ratio in -1,3-galactosyltransferase (GalT) knock-out (KO) transgenic pigs according to generation. GalT KO piglets were produced by artificial insemination or natural mating. The transgenic confirmation of GalT KO was evaluated by PCR amplification using specific primers. After electrophoresis, three types of bands were detected such as 2.3 kb single band (Wild), 2.3 and 3.6kb double bands (GalT KO -/+; heterozygote), and 3.6kb single band (GalT KO -/-; homozygote). Transgenic efficiency in F1 generation was 64.5% (23/35) of GalT KO (-/+). In F2 generation, GalT KO transgenic efficiency was 36.4% (21/57, Wild), 47.5% (28/57, GalT KO -/+), and 16.1% (8/57, GalT KO -/-), respectively. Interestingly, no homozygote piglets were born in 6 deliveries among total 11 deliveries, although they were pregnant between male (M) and female (F) heterozygote. In the 5 litters including at least one GalT KO -/- piglet, the transgenic efficiency was 13.3% (2/24, Wild), 51.3% (14/24, GalT KO -/+), and 35.3% (8/24, GalT KO -/-), respectively. The sex ratio of M and F was 40:60 in and 49:51 in generation, respectively. Based on these results, GalT KO transgenic pigs have had a reproductive ability with a normal range of transgenic efficiency and sex ratio.

Although the National Institute of Health (NIH, USA) miniature pigs were developed specifically for xenotransplantation, the cloning efficiency is still very low. To increase the efficiency, an advanced somatic cell nuclear transfer (SCNT) method may need. In the present study, we report the productions of genetically modified cloned pigs using the frozen-thawed donor cells without culture before SCNT. Fibroblasts were isolated from an ear skin of a 10-day-old NIH miniature pig. The fibroblast cells were genetically modified with the human CD73 (hCD73). For SCNT, somatic cells transfected with hCD73 were used as donor cells. The survival rate of the somatic cells was significantly higher in 0 h (95%) compared with 1 h (81%) after thawing (p<0.05). We obtained the pregnancy (38.9%, 7/18) and delivery (11.1%, 2/18) rate, respectively. Totally 7 genetically modified cloned piglets were delivered. Among them, 2 piglets were survived and 5 piglets were born stillbirth. The healthy 2 piglets are still survived (≥6 months).

Pig‐to‐human transplantation (xenotransplantation) is currently the most advanced approach to solving the increasing demand for human organs and tissues. However, two critical requirements must be addressed before xenotransplantation can be considered for clinical application. First, the level of immunosuppression required to maintain xenografts must be equivalent to (or less than) that used in allotransplantation. It is now evident that multiple genetic modifications of the donor pig will be needed to achieve this goal (d’Apice et al. 2002 Transplant Proceedings. 33: 3053‐3054). These include gene knockouts (e.g. of the GalT gene, responsible for synthesis of the major porcine xenoantigen) and gene addition by transgenesis. Progress has been hindered by the current technology, which allows only a single cycle of genetic modification per generation and therefore necessitates large and complex breeding programs. Second, donor pigs should have defined, relatively homogeneous genotypes including the inability to produce endogenous retroviruses (PERV) that may infect human recipients. Inbred miniature swine are best suited in this regard but are difficult to genetically manipulate due to poor reproductive capacity. What is critically needed to advance xenotransplantation to the clinic is the ability to perform multiple cycles of genetic modifications per generation on the background of choice. We have recently made an important step towards this goal by developing a novel method for the isolation of porcine embryonic stem cells (ESC) (Vassiliev et al. 2010 Cellular Reprogramming 12: 223‐230). These cells can be stably grown for at least 150 population doublings, dramatically increasing the window for introducing multiple genetic modifications before the cells are used to clone pigs by somatic cell nuclear transfer (SCNT). Furthermore we have used this method to isolate ESCs from cloned embryos (Vassiliev et al 2011 Cellular Reprogramming 13: 205‐213) which allows us to isolate ESCs directly from breeds of pigs specifically bred for xenotransplantation. Together these advances will accelerate xenotransplantation research to the clinic.

The present study investigated the physiological evaluation of cloned mini-pigs in a transportable isolator. Transportable isolator was designed and manufactured by our research team for transporting gnotobiotic pig. Until now, no previous reports are available regarding the physiological activities and harmful effects when pigs were transported in this isolator. Five cloned mini-pigs of 1~2 year (s) old female with a body weight between 80~90 kg were used. The effects of transportable isolator on stress-related hormone, adrenocorticotrophic hormone (ACTH) and cortisol levels, and heart rate were evaluated. In addition, it was also examined the effects of transportable isolator on blood chemistry factors (alanine aminotransferase: ALT, aspartate aminotransferase: AST, blood urea nitrogen: BUN, glucose, and creatinine). Blood was sampled just before the beginning of transport (T0), at the end of transport (30min after the transport; T1), and 30 min after the end of transport (T2). At the same time, heart rate was also evaluated. As a result, heart rate had no significant (p>0.05) differences at the various-time points of study (T0, T1, T2). However, heart rate was slightly higher than normal range in T1 and T2. The ACTH level was higher than normal range. Whereas, the cortisol level was lower than normal range. There were no statistical significant differences both ACTH and cortisol level between different time groups. Also, there were no significant differences in blood chemistry factors. Therefore, our present study shows that transportable isolator has no harmful effect on stress and physiological condition in cloned mini-pigs.