Several human leukocyte subsets including natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and polymorphonuclear neutrophils (PMN) participate in cellular immune responses directed against vascularized pig-to-human xenografts. As these leukocytes express the death receptor Fas either constitutively (PMN) or upon activation (NK, CTL), we explored in vitro whether the transgenic expression of membrane-bound human Fas ligand (mFasL) on porcine fetal fibroblasts is a valuable strategy to protect porcine xenografts. cDNA of mFasL carrying the deletion at the cleavage site with metalloproteinase and lacking the death domain in its cytoplasmic tail was subcloned into pCAGGS expression vector driven by the chicken β-actin promoter containing blastidin- resistance cassette. The mFasL expression vector was transfected into mini-pig fetal fibroblasts by lipofection method. Blastidin-resistant cells were screened by PCR and FISH. The expression of mFasL was confirmed by Western blot and FACS with the mouse anti-human FasL antibody. Interaction of two transgenic clonal cell lines with human leukocytes was analyzed using functional assay for cytotoxicity. mFasL expressed on porcine fetal fibroblasts protected porcine fetal fibroblasts against killing mediated by human NK cells. The rate of NK cell mediated cytotoxicity was significantly reduced in transgenic clonal cells (54±10.80%) compared to normal minipig fetal fibroblasts. This result indicated that grafts of transgenic pigs expressing mFasL could control the cellular immune response to xenografts, and create a window of opportunity to facilitate xenograft survival.

Unstable Epigenetic reprogramming was DNA methylation, imprinting, RNA silencing, co-valent modifications of histones and remodelling by other chromatin-associated complexes. After fusion with an enucleated oocyte, a differentiated somatic cell can restructure its genetic program and acquire totipotent characteristics. However, these cases happen only with low frequency. primordial germ cells (PGC) was effectively remove of epigenetic modifications in the genetic totipotency which is necessary for the development. The present study was in vitro development of reconstruct PGC NT embryos compared with somatic cell NT embryos. The rate of cleavage did not differ between NT embryos from PGC and somatic cells (87.26 vs 91.36%). However, the rate of development to the blastocyst stage was significantly higher in PGC cell NT than somatic cell NT (31.03 vs 19.27%). PGC from a slightly younger stage of development have succeed to promote normal development of recipient eggs. This difference in results between germ cell and somatic cell nuclear transfers could only be a reflection of intimate differences in their reprograming. These results suggest that PGC NT embryos are significantly higher for the in vitro development. Furthermore, These study may represent an approach towards achieving better production of transgenic animal.

The necessity of conditional gene expression in pigs for transgenic models is raised. Thus, in this study, Cre-loxP conditional expression in porcine fetal fibroblasts was investigated and the transformed fibroblasts were reprogrammed in enucleated oocytes for further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with pCALNL-DsRed including floxed neomycin resistant gene and selected with 750 ug/mL neomycin for two weeks. The transfected cells did not express DsRed under fluorescence microscope. After transient transfection of plasmid DNA expressing Cre, the fibroblasts began to express DsRed. The cells expressing Ds- Red were employed into somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%)were cleaved. Six blastocysts were grown up after SCNT and expressed DsRed. Deletion of floxed neomycin resistant gene was confirmed by RT-PCR in cloned blastocysts. Taken together, this study demonstrated that Cre-loxP recombination in miniature pig fibroblasts were successfully worked and those sequential transformed cells were developed into pre-implantation stage via SCNT.

Some tissues retain extensive regeneration potential through out adult life and remain as active sites of cell production. Various cell types present in tissues are being produced through proliferation and progressive specialization from a pool of stem cells. In this regard, adult stem cells (ASCs) are multipotent progenitor cells with an ability to proliferate in vitro and undergo extensive self-renewal and differentiation into a wide range of cell types, including adipocytes, chondrocytes, osteocytes, myocytes, cardiomyocytes and neurons. In addition, recent studies showing the abilities of ASCs in generating oocytes-like cells (OLCs) present new perspectives to understand the specification and interaction during the germ cell formation and oogenesis. In the present study, ASCs were established from skin, adipose and ovarian tissues of minipigs. Isolated cells exhibited a fibroblast-like morphology with higher proliferation potential and stronger alkaline phosphatase (AP) activity. ASCs from all tissues expressed pluripotent transcriptional factors, such as Oct-3/4, Nanog and Sox-2 and phenotypic markers, including CD29, CD44, CD90 and vimentin. Further, ASCs were successfully dIfferentiated into osteocytes, adipocytes and neuron-like cells. Upon induction in oogenesis specific media, all ASCs were capable of differentiation into OLCs by exhibiting distinct morphological features. Generated OLCs expressed a range of germ cell specific markers, such as Vasa, deleted in Azoospermia-like (DAZL) factor, stella, c-kit, c-Mos, synaptonemal complex protein 3 (SCP-3), growth differentiation factor 9b (GDF- 9b), zona pellucida C (ZPC) and follicle stimulating hormone receptor (FSHR) at different time points of induction. Differentiated OLCs were also positive for the expression of Vasa and DAZL protein markers. Our findings showing that OLCs can be generated from ASCs of different tissue origin may offer pig as a suitable model for designing transgenic application strategies for reproductive tissue therapy. However, further studies are needed to understand the cellular and molecular mechanisms involved in germ cell differentiation from tissue specific stem cells.

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.

Aujeszky's disease (AD), also called pseudorabies, is an infectious viral disease caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. Aujeszky's disease virus (ADV) virions contain several envelope glycoproteins. Among them, glycoproteins gB, gC and gD are regarded as the major immunogenicity proteins and the antibodies induced by them can neutralize virus in vitro or in vivo. In this study, we investigated expression of these glycoproteins using the bacterial and baculovirus expressionn system. Successful expression of ADV glycoproteins in E. coli was confirmed by SDS-PAGE and Western blot analysis and their optimal expression condition was determined. However, the recombinant proteins generated in the bacterial expression system which lacks glycosylation process frequently lose their biological activity. We tried to express the ADV glycoproteins using the baculovirus expression vector system. The recombinant gB, gC and gD were detected at approximately 100, 60 and 50 kDa on SDS-PAGE and Western blotting, respectively. The optimal expression conditions were determined for MOI(multiplicity of infection) and post-infection days. One MOI and 4 or 5 days post-infection were the best conditions for the expression of the ADV glycoproteins in Sf21 cells. We are currently investigating the antigenicity of recombinant proteins using experimental animals.

The pig has been considered to serve as an appropriate model of human disease. Therefore, establishment of porcine embryonic stem cell lines is important. The purpose of the present study was to further work in this direction. We produced porcine parthenogenetic embryos, and separately aggregated two of each of two-cell (2×2), four-cell (2×4), and eight-cell (2×8) embryos derived by parthenogenesis. After culture for 4 days, the developmental ability of the aggregates and total blastocyst cell numbers were evaluated. The percentage of blastocysts was significantly higher in both 2×4- and 2×8-aggregated embryos (58.3±1.9% and 37.2±2.8%, respectively) than in the control or 2×2-aggregated embryos (23.6±1.1% and 12.5±2.4%, respectively). Total blastocyst cell numbers were increased in the 2×4- and 2×8-aggregated embryos (by 44±3.0% and 45±3.3%, respectively) compared with those of control or 2×2-aggregated embryos (30.5±2.1% and 30.7±2.6%, respectively; p<0.05). The levels of mRNA encoding Oct-4 were higher in both the 2×4- and 2×8-aggregated embryos than in the control. When blastocysts derived from 2×4- aggregated embryos or intact normal embryos were cultured on mouse embryonic fibroblast feeder cells to obtain porcine stem cells, blastocysts from aggregated embryos formed colonies that were better in shape compared with those derived from intact blastocysts. Together, the data show that aggregation of porcine embryos not only improves blastocyst quality but also serves as an efficient procedure by which porcine embryonic stem cells can become established.

Nanotechnology is currently receiving considerable attention in various fields of biotechnology. The uptake of nanoparticles by cells for labeling and tracking is a critical process for many biomedical therapeutic applications. However, nanoparticle labeling of porcine hematopoietic cells has not been demonstrated so far. In the present study, silica-coated nanoparticles conjugated with rhodamine B isothiocyanate (SR-RITC) were used to investigate the uptake of nanoparticles by porcine hematopoietic cells. Flow cytometric and confocal microscopic analyses reveled that the cells were efficiently internalized by the silica-coated nanoparticles. Furthermore, biocompatibility tests demonstrated that the SR nanoparticles were not cytotoxic, and they had no impact on proliferation. Our study demonstrates that silica-coated nanoparticles are taken up very rapidly and with high efficiency into porcine hematopoietic cells, with no apparent deleterious effects. Therefore, silica-coated nanoparticles appear to be a promising tool for tracking porcine hematopoietic cells.

Plasminogen activators (PAs) are serine proteases that convert plasminogen to plasmin. The PA/plasmin system has been associated with a number of physiological processes such as fibrinolysis, ovulation and fertilization. Although correlations have been reported between reactive oxygen species (ROS) and oocyte maturation, the relationship between PA activity and ROS is unknown. The present study was undertaken to determine the effects of cumulus cells on PA activity in matured porcine oocytes under xanthine (X)-xanthine oxidase (XO) system. When oocytes were matured under the X-XO system, the proportion of oocytes remaining GV stage was higher (p<0.05) in oocytes without cumulus cells. The incidence of degenerated oocytes was higher (p<0.05) in the X+XO ( and ) than in the control group ( and ). The proportion of TUNEL-positive oocytes and activity of caspase-3 were higher (p<0.05) in cumulus-free oocytes and oocytes exposed to ROS. Tissue-type plasminogen activator-plasminogen activator inhibitor (tPA-PAI) and tissue-type plasminogen activator (tPA) activity were detected in oocytes that were separated from cumulus-oocytes complexs (COCs) at 44 h of maturation culture, and only tPA was produced in oocytes that were denuded before the onset of maturation culture. On the other hand, the activities of PA were increased (p<0.05) when oocytes were cultured under the X-XO system. The higher activity of tPA was observed in denuded oocytes (DOs) underwent apoptotic changes by oxidative stress. In COCs, however, tPA-PAI as well as tPA activity was detected and apoptotic changes such as DNA cleavage or caspase-3 activation were not observed. These results suggest that tP A may be relevant to apoptotic cell death in porcine oocytes by oxidative stress.

To examine the differential expression of proteins during the cycling (70～80% confluences) and G0/G1 (full confluences) phases in porcine fetal fibroblast cells, we used a global proteomics approach by 2‐D gel electrophoresis (2‐DE) and MALDI‐TOF‐MS. Cycling cell were harvested at approximately 70% to 80% confluent state while cells in G0/G1 phase were recovered after maintenance of a confluent state for 48 hr. Cellular proteins with isoelectric points ranging between 3.0～10.0, were analyzed by 2‐DE with 2 replicates of each sample. A total of approximately 700 spots were detected by 2‐D gels stained with Coomassie brilliant blue. On comparing the cell samples obtained from the cycling and G0/G1 phases, a total of 13 spots were identified as differentially expressed proteins, of which 8 spots were up‐regulated in the cycling cell and 5 were up‐regulated in the G0/G1 phase. Differentially expressed proteins included K3 keratin, similar to serine protease 23 precursor, protein disulfide‐isomerase A3, microsomal protease ER‐60, alpha‐actinin‐2, and heat‐shock protein 90 beta. The identified proteins were grouped on the basis of their basic functions such as molecular binding, catabolic, cell growth, and transcription regulatory proteins. Our results show expression profiles of key proteins in porcine fetal fibroblast cells during different cell cycle status.

The present study was performed to identify changes of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) in porcine oviduct epithelial cells (POECs) during the estrous cycle. POECs obtained from ovary in pre‐ovulatory (Pre‐Ov), early to mid‐luteal stage (Early‐mid L) and post‐ovulatory stage (Post‐Ov). For the examine of PA activity, 1×105 fresh cells of POECs were cultured in DMEM/Ham F‐12 containing 10% FBS and 0.2% amphotericin under humidified atmosphere of 5% CO2 in air and 38℃. The urokinase‐type PA (uPA) was observed at 7 days of POECs culture. PA activity was measured with culture prolonged of 0, 3, 6, 12 and 24 hafter culture of 7 days. The PA activity were high significantly (p<0.05) at 12 h of culture, but PA activity were decreased with culture periods increased. The PA activity in POECs of Post‐Ov stage were higher significantly (p<0.05) than that of Early‐mid L and Pre‐Ov stage. When PAI‐1 and PAI‐2 were added during the POECs culture, the PA were observed significant low activity (p<0.05). The PA activity and protein expression were decreased by PA inhibitor. This results suggest that PAI‐1 and PAI‐2 have a suppressive action on change of PA activity uring the estrous cycle of pigs. Specifically, this study using PA inhibitor was effect the PA activity and PAI expression in oviduct epithelial cells in pigs.

Spermatogonial stem cells(SSCs) only are responsible for the generation of progeny and for the transmission of genetic information to the next generation in male. Other in vitro studies have cultured SSCs for proliferation, differentiation, and genetic modification in mouse and rat. Currently, information regarding in vitro culture of porcine Germline Stem Cell(GSC) such as gonocyte or SSC is limited and is in need of further studies. Therefore, in this study, we report development of a successful culture system for gonocytes of neonatal porcine testes. Testis cells were extracted from 10～14-day-old pigs. These cells were harvested using enzymatic digestion, and the harvested cells were purified with combination of percoll, laminin, and gelatin selection techniques. The most effective culture system of porcine gonocytes was established through trial experiments which made a comparison between different feeder cells, medium, serum concentrations, temperatures, and O2 tensions. Taken together, the optimal condition was established using C166 or Mouse Embryonic Fibroblast(MEF) feeder cell, Rat Serum Free Medium(RSFM), 0% serum concentration, 37℃ temperature, and O2 20% tension. Although we discovered the optimal culture condition for proliferation of porcine gonocytes, the gonocyte colonies ceased to expand after one month. These results suggest inadequate acquirement of ingredients essential for long term culture of porcine GSCs. Consequently, further study should be conducted to establish a successful long-term culture system for porcine GSCs by introducing various growth factors or nutrients.