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.

Poly(ADP-ribosyl)ation is post-translational modification of cellular proteins related to cell survival, cell death, cellular proliferation and epigenetic events. It has recently been shown to be important for pre-implantation development of mouse embryos. However, its function during early embryonic development of pig is not clear. This study investigated the importance of poly(ADP-ribosyl)ation during in vitro development of pig embryos produced by in vitro fertilization(IVF) or parthenogenetic activation (PA). Results showed that, chemical inhibition of PARP by 3-aminobenzamide (3-AB) did not influence the in vitro development of pig embryos up to morula stage (20±3.1 vs. 28.1±1.2%; p>0.05) but significanlty reduced the rate of blastocyst formation (5.2±2.1 vs. 20±3.1%; p<0.05) when compared to non-treated controls. Furthermore, culture of morula stage embryos in the pressence of 3-AB for 24h significantly reduced the rate of blastocyst formation (19.6± 4.6 vs. 41.4±5.3%; p<0.05) and expansion (4.7±3.0 vs. 28.1±6.1; p<0.05). The proportion of large-sized blastocyst (>200 μm) having higher blastocoel volume (15.3×106 μm3) was significantly reduced (p<0.05) in treatment group (32.2±7.8%) compared to non-treated control group (65.7±9.0%). TUNEL assay revealed that poly(ADP-ribosyl)ation-inhibited blastocyst had significantly increased indices of apoptosis than those of non-treated controls (10.88±0.02 vs. 2.71±0.01; p<0.05). These data suggest that Poly(ADP-ribosyl)ation may be important for blastocyst formation in pig embryo.

Urokinas type plasminogen activator (uPA) has been used as a therapeutic agent for treating human diseases such as thrombosis. Attempts to transgenically overexpress the uPA in animal bioreactors have been hampered due to side effects associated with this functional protein hormone on homeostasis. Recently, chicken has been emerged as a potential candidate for use as bioreactor to produce proteins of pharmaceutical importance. Since this species has low homology uPA sequence with mammals, we hypothesized that chicken could be used as a potential bioreactor for production of human uPA. In this study, using replication‐defective Murine Leukemia Virus (MLV)‐based retrovirus vectors encapsidated with Vesicular Stomatitis Virus G Glycoprotein (VSV‐G), we attempted to make transgenic chicken expressing human uPA (huPA). The recombinant retrovirus was injected beneath the blastoderm of non‐incubated chicken embryos (stage X, at laying). After 21 days of incubation (at hatching), all of the 38 living chicks that assayed, were found to express the vector‐encoded huPA gene in various organs and tissues, which was under the control of the Rous Sarcoma Virus (RSV) or Cytomegalovirus (CMV) promoter. Using specific primer set for huPA, PCR and RTPCR analyses of gDNA isolated from these samples demonstrated these chickens were transgenic for huPA. Furthermore, successful germ line transmission of huPA transgene was confirmed and next generation whole body huPA transgenic chickens were also produced. We also assayed huPA protein titer in blood (17.1 IU/ml) and eggs (4.4 IU/ml) of whole body huPA transgenic chicken. Thus, our results demonstrated that chicken could be used as bioreactors to produce huPA.

Testes‐derived unipotent male germ‐line stem (GS) cells can acquire multipotency under appropriate culture conditions to become mGS cells which can contribute to all three germ‐layers. This study was designed to investigate the epigenetic characteristics of mGS cells derived from adult mouse testes (maGS cells). The GS cells were isolated from 4 6 week DBA mouse and were cultured in Dulbecco’s modified Eagle Medium supplemented with 15% (v/v) fetal bovine serum, 1,000 U/ml LIF, 4 ng/ml GDNF at 37℃ in an humidified atmosphere of 5% CO2 in air to derive the maGS cells. The multipotency of maGS cells were verified by morphological and gene expression analyses, teratoma formation upon transplantation into nude mouse and in vitro differentiation ability. Bisulfite genomic sequencing revealed that GS cells had androgenetic DNA methylation pattern at the Igf2‐H19, Gnas‐Nespas , and Dlk1‐Dio3 imprinted gene clusters which changed to hemi‐zygotic embryonic stem (ES)‐cell like pattern in the maGS cells. Western blot analysis, using modification‐ and residue‐specific antibodies, revealed that both maGS and ES cells had similar level of histone di‐methylation at 4th and 27th lysine residue of histone 3 (H3K4me2 and H3K27me2) which represent “bivalent domain” for regulating self‐renewal and differentiation of mouse ES cells. Both maGS and ES cells also shared similar hisone modification for H3K9me2, H3K79me2, H3K9ac and H3K18ac. However, maGS cells had higher level of H3K- 36me2 and H3S10p. These data suggest that maGS and ES cells share several epigenetic characteristics but they also have their own unique epigenetic marks that may be useful as a molecular marker for their identification.

Preservation of sperm is essential for long-term storage of valuable animal genetic resources and for the conservation of threatened mammalian species undergoing progressive extinction. In this study, using pig as a model system, we evaluated the feasibility of germ-plasm preservation via sperm cell lyophilization. We show that, pig sperm can be successfully lyophilized and stored in a liquid nitrogen-free condition for at least 6 months. Intracytoplasmic injection of lyophilized sperm (ICSI), stored at 4℃ for four months, into in vitro matured pig oocytes could successfully develop up to blastocyst stage (13.0±3.0%). Lyophilized sperm could also be stored at room temperature for at least three weeks without further compromising their in vitro development up to the blastocyst stage (14.6±3.2 vs. 16.6±5.1%; p>0.05). Blastocysts produced from ICSI of lyophilized sperm stored at 4℃ or room temperature contained similar number of cells per blastocyst (44.9±3.2 vs. 44.0±4.3; p>0.05) but was significantly lower than those produced from non-lyophilized fresh sperm (52.1±5.8 p>0.05). Interestingly, use of a custom-designed HEPES-buffered, calcium-free, defined medium for the lyophilization resulted in normal post-ICSI embryonic development up to blastula stage (23.4±2.8 vs. 24.0±2.9%) and, the resultant blastocysts contained similar number of cells per blastocyst (47.9±4.3 vs. 50.6±7.0) compared to those generated from non-lyophilized fresh sperm (p>0.05). These lyophilized sperm could also be stored at room temperature for at least three weeks with slight reduction in post-ICSI embryonic development (19.6±1.4%). Therefore, these results suggest that, pig sperm could be successfully and efficiently lyophilized for their long-term storage at 4℃. Lyophilization of sperm could be a practical option for long-term storage of mammalian germ-plasm.

Autophagy, the process of bulk degradation and recycling of long-lived proteins, macromolecular aggregates, and damaged intracellular organelles, has recently been shown to be important for pre-implantation development and cavitation in mouse embryos. This study investigated the occurrence of autophagy and its importance in determining the in vitro development of pig embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). Western blot analysis for autophagy marker, microtubule associated protein light chain 3 (MAP-LC3), revealed the temporal pattern of LC3-conversion with intense changes during 10 20 h post-insemination and at morula-blastocyst transition in pig embryos. Specific inhibition of autophagy in 2 4 cell stage pig embryos, by treatment with 3-methyladenine (3MA), did not affect their embryonic development up to morula stage (p>0.05) but completely blocked their progression to the blastocyst stage (0.0±0.0 vs. 28.5±1.7% p<0.05). On the other hand, autophagy-inhibition in morula stage embryos significantly inhibited the formation of blastocoel (14.9±3.6 vs. 37.5±7.2%) and reduced the proportion of expanded blastocysts (5.6±2.6 vs. 29.6± 4.6% p<0.05). TUNEL assay revealed that autophagy-inhibited embryos had significantly increased indices of apoptosis (10.2±0.4 vs. 2.3±0.2) and DNA fragmentation (0.8± 0.1 vs. 0.3±0.1) than those of controls (p<0.05). Interestingly, while anti-oxidants reduced (p<0.05) the apoptosis and improved the blastocyst formation rate in pig embryos, it had no influence (p>0.05) on the expression of MAP-LC3. These data therefore, suggest that autophagy may have essential role during blastocyst formation in pig embryos.

The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFRα-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs (11.2±0.8%) and SSCs (13.3±1.1%). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.