Xenotransplantation of pig organs into primates results in fatal damage, referred as hyperacute rejection (HAR), and acute humoral xenograft rejection (AHXR), to the organ graft mediated by antibodies pre-existing and newly-producing in primates against their cognate pig antigens. Functional ablation of α1,3-galactosyltransferase (Gal-T KO) of pig which is an enzyme involved in synthesis of Gala1-3Galb1-4GlcNAc-R antigen is essentially required to prevent HAR. Moreover, additional genetic modification under Gal-T KO background for enforced expression of human complement regulatory proteins which can inhibits complement activation is known to effectively imped HAR and AHXR. In this study, we constructed a membrane cofactor protein (MCP) expression cassette under control of human EF1α promoter. This cassette was inserted between homologous recombination regions corresponding to Gal-T locus. Subsequently this vector was introduced into ear skin fibroblasts of female pig by nucleofection. We were able to obtained 40 clones by neomycin selection and 4 clones among them were identified as clones targeted into Gal-T locus of MCP expression cassette by long-range PCR. Real time RT-PCR was shown to down-regulation of Gal-T expression. From these results, we demonstrated human EF1α promoter could induce efficient expression of MCP on cell surface of fibroblasts of female pig.

A major barrier to progress in pig to primate organ transplantation or cell therapy is the presence of terminal α -1,3-galactosyl epitopes on the surface of pig cells. Therefore, the purpose of this experiment was to establish and cha- racterize mesenchymal stromal/stem cells (MSCs) derived from α-1,3-galactosyltransferase (GalT) knock out (GalT KO) pig to confirm their potential for cell therapy. Bone marrow (BM)-MSCs from GalT KO pig of 1 month old were isolated by Ficoll-Paque PLUS gradient and cultured with A-DMEM + 10% FBS on plastic dishes in 5% CO2 incubator at 38.5. GalT KO BM-MSCs were analyzed for the expression of CD markers (CD45－, 29+, 90+ and 105+) and in vitro differentiation ability (adiopogenesis and osteogenesis). Further, cell proliferation capacity and cell aging of GalT KO BM-MSCs were compared to Wild BM-MSCs by BrdU incorporation assay (Roche, Germany) using ELISA at intervals of two days for 7 days. Finally, the cell size was also evaluated in GalT KO and Wild BM-MSCs. Statistical analysis was performed by T-test (P<0.05). GalT KO BM-MSCs showed fibroblast-like cell morphology on plastic culture dish at passage 1 and exhibited CD45－, 29+, 90+ and 105+ expression profile. Follow in ginduction in StemPro adipogenesis and osteogenesis media for 3 weeks, GalT KO BM-MSCs were differentiated into adipocytes, as demonstrated by Oilred Ostaining of lipid vacuoles and osteocytes, as confirmed by Alizarinred Sstaining of mineral dispositions, respectively. BrdU incorporation assay showed a significant decrease in cell proliferation capacity of GalT KO BM-MSCs compared to Wild BM-MSCs from 3 day, when they were seeded at 1×103 cells/well in 96-well plate. Passage 3 GalT KO and Wild BM-MSCs at 80% confluence in culture dish were allowed to form single cells to calculate cell size. The results showed that GalT KO BM-MSCs (15.0 ± 0.4 μm) had a little larger cell size than Wild BM-MSCs (13.5 ± 0.3 μm). From the above findings, it is summarized that GalT KO BM-MSCs possessed similar biological properties with Wild BM-MSCs, but exhibited a weak cell proliferation ability and resistance to cell aging. Therefore, GalT KO BM-MSCs might form a good source for cell therapy after due consideration to low proliferation potency in vitro.

The present study was performed to investigate the effect of Hh-Ag1.5, a small-molecule chemical agonist of SMOothened receptor, on the in vitro maturation and development of in vitro fertilized (IVF) embryos in pigs. Oocytes or fertilized embryos were cultured in a maturation or embryo culture medium supplemented with 0 (control), 25, 50 or 100 nM of Hh-Ag1.5, respectively. Although the maturation rate were not different among treatment groups, the blastocyst formation rate in the group treated with 25 nM Hh-Ag1.5 was significantly increased compared to other groups (P<0.05). While the highest dose of Hh-Ag1.5 (100 nM) did negatively affect to the embryo development and cell number in blastocysts compared to other groups (P<0.05), the apoptotic cell index in blastocysts was significantly lower in 25 and 50 nM groups than in control and 100 nM groups (P<0.05). The mRNA expression of the proapoptotic gene Bax and the ratio of Bax/Bcl-XL decreased in among treatment groups compared to control (P<0.05). The embryo quality related genes, Tert and Zfp42, were significantly decreased in 50 and 100 nM groups compared with control and 25 nM groups (P<0.05). In conclusion, the addition of 25 nM Hh-Ag1.5 to in vitro maturation and culture medium can enhance the developmental potential as well as quality of IVF embryos in pig.

This study aimed at investigating whether a porcine follicular fluid (pFF) supplementation positively affects the characteristics of donor cells and the developmental competence of porcine cloned embryos. Ear fibroblast cells (donor cell) from an Massachusetts General Hospital miniature pig were cultured in different culture methods: (1) Dulbecco's modified Eagle's medium (DMEM)+10% FBS (Control); (2) DMEM+0.5% FBS (SS); and (3) DMEM+10% FBS+10% pFF (pFF) for 72 h. In each conditioned medium, the concentrations of 4 amino acids (Thr, Glu, Pro, and Val) in the pFF group were significantly different from those in the control group (p<0.05 or p<0.01). The proliferation of the cells cultured in the SS group was significantly lower than that of the other treatment groups (p<0.01). The population of apoptotic and necrotic cells in the SS group was significantly higher than that of either the control or the pFF group (p<0.01). The number of embryos that cleaved (p<0.05) and developed into blastocysts (p<0.01) in the SS group was significantly lower than that of either the control or the pFF group. Compared to other groups, the blastocysts produced from the donor cells in the pFF group had higher total cells and lower apoptotic cells (p<0.05). It can be concluded that pFF supplementation in the donor cell culture medium positively affects cell death, cell cycle and quality of the cloned blastocyst.

The shortage of human organs for transplantation has induced the research on the possibility of using animal as porcine. However, pig to human transplantation as known as xeno-transplantation has major problem as immunorejection. Recently, the solutions of pig to human xenotransplantation are commonly mentioned as having a genetically modification which include alpha 1, 3 galatosyl transferase knockout (GTKO) and immune-suppressing gene transgenic model. Unfortunately, the expression level of transgenic gene is very low activity. Therefore, development of gene overexpression system is the most urgent issue. Also, the tissue specific overexpression system is very important. Because most blood vessels are endothelial cells, establishment of the endothelial-specific promoter is attractive candidates for the introduction of suppressing immunorejection. In this study, we focus the ICAM2 promoter which has endothelial-specific regulatory region. To detect the regulatory region of ICAM2 promoter, we cloned 3.7 kb size mini-pig ICAM2 promoter. We conduct serial deletion of 5' flanking region of mini-pig ICAM2 promoter then selected promoter size as 1 kb, 1.5 kb, 2 kb, 2.5 kb, and 3 kb. To analyze promoter activity, luciferase assay system was conducted among these vectors and compare endothelial activity with epithelial cells. The reporter gene assay revealed that ICAM2 promoter has critical activity in endothelial cells (CPAE) and 1 kb size of ICAM2 promoter activity was significantly increased. Taken together, our studies suggest that mini-pig ICMA2 promoter is endothelial cell specific overexpression promoter and among above all size of promoters, 1 kb size promoter is optimal candidate to overcome the vascular immunorejection in pig to human xenotransplantation.

Genetic modification of the pig of which the gene is relevant to human diseases allows the pig to be used as a source of biomedical animal model. The promoter which could drive efficient expression constitutively or specifically of the interest gene in porcine organs is essentially required to increase versatility of a biomedical porcine model. In this study, we compared different promoters of activities driving efficient expression in different types of porcine cells including primary fibroblasts, kidney-derived PK-15, and primary endothelial cells (EC). To this end, we inserted CMV, EF1-α, CMV/EF1-α, CAG, human and porcine membrane cofactor protein gene promoters(MCP and Mcp), and porcine intercellular adhesion molecule-2 (Icam-2) promoter into pGL3 basic vector. Luciferase assay revealed that CAG promoter led to highest promoter activity in fibroblasts and PK-15 cells. CMV, EF1-α, CMV/EF1-α promoters showed moderate activities for luciferase expression in fibroblasts and PK-15 cells. Interestingly, CMV/EF1-α promoter, in which CMV promoter was linked to the front of EF1-α promoter as an enhancer led to highest luciferase expression in EC. The MCP, Mcp and Icam-2 promoters showed very low level of luciferase expression in three types of cells. Taken together, this study demonstrated that promoter activity in different porcine cells is differently expressed.

Here we report the productions of genetically modified cloned Massachusetts General Hospital miniature pig (MGH minipig) using freshly thawed donor cells equilibrated with roscovitine. Fibroblasts were isolated from the ear skin of a 10-day-old male MGH minipig. The donor cells were divided into two groups: cultured for 3 days (culture) and freshly thawed with 500 nM roscovitine. The viability of the donor cells was significantly higher at 0 h (94.6±3.5) compared with 1 h (81.7±5.7) after thawing (p=0.028). After 1 hr of equilibration time, the proportion of G0/G1 stage in roscovitine group was not different from 0 hr group, but not in culture medium group (p<0.01), respectively. Although the developmental characteristics were not different in both methods, the pregnancy and delivery rate in freshly thawed group were significantly higher than that of culture group (p<0.01), respectively. In total, 12 TG cloned MGH minipigs were delivered and the individual cloning efficiency was from 0 to 2.54%. Taken together, the use of freshly thawed donor cells equilibrated with roscovitine may be helpful method to increase the productivity of the genetically modified cloned MGH minipigs.

Pig parthenotes were able to develop in vivo for 30 days with normal morphology. In pig, during blastocyst elongation between day 10 and 12 of gestation, estrogen production and secretion by conceptus increases, serving not only as the signal for maternal recognition of pregnancy, but also as a stimulus for the production of proteins and growth factors within the uterine environment that initiate implantation. Cloning efficiency is still very low regardless of species. To increase the productive efficiency of (transgenic, TG) clones, an advanced somatic cell nuclear transfer (SCNT) method may need. Here we report the productions of transgenic cloned pigs using cloned embryos and parthenotes simultaneously. Fibroblasts were isolated from an ear skin of a 10‐day‐old NIH miniature pig. The ear fibroblast cells were transfected with the alpha1,3‐ Galactosyltransferase knock‐out/human CD46 knock‐in (GalT KO/hCD46 KI). For SCNT, the TG somatic cells were used as donor cells. Immediately after fusion confirmation, the TG cloned embryos and parthenotes were transferred into both oviducts of surrogates. The mean number of TG cloned embryos and parthenotes was 137 (±15.2) and 123(±27.1), respectively. The pregnancy and delivery rate was (55.6%, 10/ 18) (44.4%, 8/18), respectively. Totally 19 GalT KO/hCD46 KI cloned piglets were delivered. Among them, 11 piglets were survived and 8 piglets were born stillbirth. The healthy 5 piglets are still survived.

Despite of the absence of hyperacute rejection and acute humoral xenograft rejection, the organ graft of the a1,3-galactosyltransferase (GalT) gene knockouted (KO) and complement regulatory protein (CRP) expressing pig into a nonhuman primate is rejected by development of a thrombotic microangiopathy and/or a consumptive coagulopathy. Thus further introduction of genes to overcome the coagulation incompatibilities between pig and primate under GalT KO/CRP genetic background has been strongly suggested. CD73 (ecto-5'-nucelotidase) is an enzyme attached via a glycosyl phosphoinositol anchor to the extracellular membrane of endothelial cells, which catalyses the hydrolysis of adenosine triphosphate to adenosine. Loss of activity of CD73 results in activation and aggregation of platelets by a reduced capacity to convert nucleotides to adenosine. In previous study, we reported generation of GalT KO fibroblasts concurrently expressing membrane cofactor protein and produced cloned pigs by nuclear transfer of the fibroblast cells (1). In this study, we constructed a vector for expression of human CD73 under control of promoter of pig Icam2 gene expressed specifically at endothelial cells. This vector was introduced into porcine fibroblasts using the nucleofection technology, by which we had forty three fibroblasts clones carrying pIcam2- CD73 vector. Somatic cell nuclear transfer resulted in generation of two transgenic piglets survived.

Immunological rejection of the organ grafted onto a primate arises from two antibody mediated processes, hyperacute rejection (HAR) and acute humoral rejection (AHR). Functional ablation of α1,3-galactosyltransferase (GalT) and concurrently overexpression of complement regulatory proteins are known to inhibit HAR and AHR. In previous study, we reported that production of porcine male fibroblasts harboring a MCP expression cassette targeted to GalT locus. In this study, we constructed a different MCP expression cassette, in which the EF1α promoter regulates MCP expression and internal ribosome entry site-mediated neomycin resistance gene expression. Subsequently, this cassette was inserted between the left and the right homologous arms to target exon 9 of the GalT gene. Female fibroblasts were isolated from ear skin of 10 days old miniature pig, and used for nucelofection of the the construct for MCP expression at GalT locus. PCR analysis showed that four clones of forty neomycin resistant clones carry MCP expression cassette at exon 9 of the GalT gene. Two clones analyzed downregulated GalT expression, as determined by quantitative reverse transcriptase polymerase chain reaction. Flow cytometry analysis showed that MCP was efficiently expressed at the cell surface.

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).

The purpose of this study was undertaken to evaluate of cryopreservation efficiency in α 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen (LN2) vapours for 10 min before placing them into LN2 for cryopreservation. A fter thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.

Fibroblasts of large animals are easy to isolate and to maintain in vitro culture. Thus, these cells are extensively applied to donor cell for somatic cell nuclear transfer, and to substrate cells to generate induced pluripotent stem cells after transfection of required genes to be essentially required for direct reprogramming. However, limited mitotic activity of fibroblasts to differentiate along a terminal lineage becomes restrictive for their versatile application. Recently, commercial culture medium and systems developed for primary cells are provided by manufactures. In this study, we examined whether one of the systems developed for primary fibroblasts of human are effective on porcine ear skin fibroblasts. To this end, we performed proliferation assay after five days culture in vitro of porcine fibroblasts in medium DMEM, which is generally used for fibroblasts culture, and medium M106 for human dermal fibroblasts, supplemented with various concentrations of FBS and LSGS contained mainly growth factors, respectively. Consequence was that presence of 15% FBS and 0.1 X concentrations of LSGS in DMEM showed most active proliferation of porcine fibroblasts.

This study was conducted to investigate the effects of donor cell treatments on the production of transgenic cloned piglets. Ear fibroblast cell obtained from NIH MHC Inbred minipig was used as control. The GalT knock-out/CD46 knock-in (GalT/CD46) transgenic cell lines were established and used as donor cells. The reconstructed GalT/CD46 embryos were surgically transferred into oviduct of naturally cycling surrogate sows (Landrace × Yorkshire) on the second day of standing estrus. Unlike control (1.2 kV/cm,, 75.4%), the fusion rate of the GalT/CD46 donor cells was significantly higher in 1.5 kV/cm, (84.5%) than that of 1.25 kV/cm, (20.2%) (p<0.01). When the number of the transferred embryos were more than 129, the pregnancy and delivery rates were increased to 13/20 (65%) and 5/20 (25%) compared to less than 100 group [1/6 (16.7%) and 0/6 (0%)], respectively. To analyze the effect of donor cell culture condition on pregnancy and delivery rates, the GalT/CD46 donor cells were cultured with DMEM or serum reduced medium. In serum reduced medium group, the pregnancy and delivery rates were improved to 8/12 (66.7%) and 5/12 (41.7%) compared to DMEM group [3/7 (42.9%) and 0/7 (0%)], respectively. In conclusion, it can be postulated that an appropriate fusion condition and culture system is essential factors to increase the efficiency of the production of transgenic cloned piglets.