The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R) is responsible for hyperacute rejection (HAR) during transgenic pig-to-non-human primate xenotransplantation. To overcome HAR after xenografts, it is essential for the inactivation of α1,3Galactosyltransferase (GT) gene by the homozygotic knocked out of GT-/- and the isoglobotrihexosylceramide synthase (iGb3s-/-). This study was performed to investigate the generation and characterization of the α1,3GT-MCP/-MCP+iGb3-/- transgenic cells. Ear fibroblast cells from the GT-MCP/-MCP pig were cultured and used to positive control. For iGb3s knock out, the Cas9-GFP-iGb3s vector was transfected into the GT-MCP/-MCP cells. The Cas9-GFP-iGb3s transfected cells were sorted and sequenced for the selection of GT-MCP/-MCP+ iGb3s-/- cells. Among the three sorted cell lines, one transgenic cell line was homozygously deleted 3 bases and 10 bases in each chromosome, respectively. To characterize an expression of α-Gal epitope, a wild type and the transgenic cells were measured by FACS Aria using BS-IB4 lectin antibody. The expression of α-Gal epitope in GT-MCP/-MCP cells (<0.01 %) were significantly down-regulated to the range of wild type (99.4 %) fibroblast cells (p<0.05). To analyze the function of iGb3s, α -Gal epitope expressions were measured for the GT-MCP/-MCP, GT-MCP/-MCP+iGb3s-/+, and GT-MCP/-MCP+iGb3s-/-. The range was 95.8%, 94.2%, and 75.8%, respectively. Interestingly, there was a negative range (16.2%) of α-Gal epitope -/- section in GT-MCP/-MCP+iGb3s-/-, compared to 2.74% of GT-MCP/-MCP+iGb3s-/+ and 1.4% of WT, respectively. Our results demonstrated that iGb3s-/-combined with GT-/- had a function to inhibit α-Gal epitope expression in pig cells. Further studies are needed to evaluate the functions of double gene knock out to minimize a HAR response after xenotransplantation.

The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R) is responsible for hyperacute rejection (HAR) during transgenic pig-to-non-human primate xenotransplantation. There are genes related to the expression of α-Gal epitope such as α1,3Galactosyltransferase gene (GT-/-) and the isoglobotrihexosylceramide synthase (iGb3s-/-). This study was performed to investigate the expression of α-Gal epitope in the skin derived from GT-/- transgenic pig. The skin (7/1000 inches) was obtained by dermatome (Zimmer® Electric Dermatome) from one month old of wildtype (WT) and GT-/- piglets, respectively. The skins were fixed, dehydrated, cleaned, and embedded. To analyze the expression of α-Gal epitope, the paraffin section of WT and GT-/- were stained with BS-IB4 lectin and isoglobotrihexosylceramide synthase antibody. There was a strong BS-IB4 lectin signal in the skin of WT, but not detected in GT-/-. However, the iGb3s positive signals were stained in the skin of both WT and GT-/-. Taken together, it can be postulated that the knocked out of GT gene may not enough to inhibit the expression of α-Gal epitope. Further studies are needed to evaluate the functions of the double knock out of GT and iGb3s on the expression of α-Gal epitope.

Human interferon alpha 2b (hIFNα-2b) is an important immune regulator widely used in clinic, for the treatment of chronic hepatitis, hairy cell leukemia, chronic myelogenous leukemia and multiple myeloma, etc. The clinically used hIFNα-2b is generally produced by E. Coli, which lacks the post-translational O-glycosylation of naturally synthesized protein, and has a short serum half-life. In this study, we report the successful generation of transgenic chickens that produce hIFNα-2b in the egg white using a feline immunodeficiency virus (FIV)-based lentiviral vector. In preliminary in vitro study, the hIFNα-2b gene under the control of CMV promoter expressed as much as 2,650 ng/㎖ in CEF-LNC-hIFNα-2bW cell. A FIV vector packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected underneath the blastoderm of freshly laid chicken eggs (stage X) to produce a hIFNα -2b transgenic chicken. Out of 187 injected eggs, 55 chicks were hatched after 21 days of incubation, and 27 of the G0 hatched chicks expressed the vector-encoded hIFNα-2b gene. The expression of recombinant hIFNα-2b in transgenic chickens constitutes an important step towards low-cost and full biological activity production of this protein drug in bioreactor. This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ011178), Rural Development Administration, Republic of Korea.

In the present study, using a MoMLV-based retrovirus vector, we successfully generated a new transgenic chicken line expressing high levels of hEPO. A replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of non-incubated chicken embryos (stage X). One rooster was mated to wild-type hens to produce 748 G1 progeny. PCR analysis of blood samples from these progeny revealed that there were seven G1 transgenic offspring, corresponding to a 0.9% germline transmission rate. Subsequently, Southern blot analysis of the genomic DNA from three G1 transgenic chickens was carried out to verify the stable genomic integration and copy number of the transgene in the genome. Quantitative analyses of the blood samples taken from G1 transgenic chickens resulted in 4,150 ～ 10,823 IU/㎖ (34.6 ～ 90.2 ㎍/㎖) of hEPO in the blood. The biological activity of the recombinant hEPO in transgenic chicken serum was comparable to its commercially available counterpart. Red blood cell numbers were more than three-fold higher in the transgenic chickens compared to the non-transgenic chickens. Successful germline transmission of the transgene was also confirmed in G2 transgenic chicks produced from crossing G1 transgenic roosters with non-transgenic hens. We confirmed that 13 transgenic chicks of 45 G2 progeny, corresponding to a 28.9% germline transmission rate. These results will help establish a useful transgenic chicken model system for studies of embryonic development and for efficient production of transgenic chickens as bioreactors. This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ011178), Rural Development Administration, Republic of Korea.

RNA Sendai virus (SeV) vector system has no risk of being integrated into the host genome. Sendai virus (SeV) vectors expressing pluripotent factors have been used to produce integration-free induced pluripotent stem cells (iPSCs) with high efficiency from various cell types in human and mouse. In this study, we generated iPSCs from pig ear fibroblast cells using the SeV vector expressing 4 human factors (POU5F1, SOX2, C-MYC, and KLF4). Colonies were emerged at Day 14 of transduction and expressed the classical pluripotency markers (POU5F1, NANOG, and SOX2) and surface marker (SSEA1). Furthermore, they showed a domed shape and could passage over 40 times under 2i (CHIR99021 and PD0325901)-LIF and MEF feeder culture condition having in vitro differentiation ability into 3 germ layers. Next, we examined the ability of six feeder free culture conditions to maintain piPSCs in a pluripotent state. piPSCs were plated on Matrigel coated dishes in different media: 1. CM: control media (LIF culture media); 2. CM-F: CM+100 ng Fetuin-A; 3. CM-N: CM+100 ng Nanog-TAT; 4. CM-2i: CM+3 uM CHIR99021+1 uM PD0325901; 5. CM-2iN: CM-2i+100 ng Nanog-TAT; 6. CM-2iN+100 ng Fetuin-A. However, piPSC could not maintain the typical self-renewal morphology on feeder free conditions regardless of culture media tested here. Further, expression of pluripotency-related genes (Oct4, Nanog and Klf4) of piPSCs cultured on feeder free conditions could not be compared with that of iPSCs cultured on MEF feeder plate. Our results suggest that integration free pluripotent stem cell from pigs could be generated by SeV vector system and maintained their pluripotency under 2i-LIF and MEF feeder culture condition, but further optimization of culture conditions may be required.

It is still challenging to establish pESCs due to differences in the genetic backgrounds of mouse, human, and pig. So it is required to find pig specific pluripotency markers and cellular signaling. In this experiments, doxycycline-inducible vectors carrying OCT4, SOX2, NANOG, KLF4 and MYC known as reprogramming factors, were infected into pig stem cells for analyzing gene expression pattern. When cultured without doxycycline, pig stem cells were stably maintained in bFGF supplemented media. However, when treated with doxycycline, pig stem cells lost alkaline phosphatase activity and were differentiated within two weeks. And then, we investigated the expression of genes related to pluripotency in doxycycline-treated pig stem cells by using qRT-PCR. The qRT-PCR data revealed that expression of OCT4, CDH1 and FUT4 were significantly increased by OCT4 overexpression and OCT4 and FUT4 were also upregulated in SOX2-infected group. When infected with combination of two factors including OCT4 or SOX2, some groups could stably maintain at LIF supplemented media, having alkaline phosphatase activity. Given these data, although ectopic gene expression induced differentiation in pig stem cells, ectopic expression of OCT4 and SOX2 could upregulate pluripotent genes and overexpreession of two factors help pig stem cells adapt LIF-contained media. This study could improve understanding of pluripotent networks as well as aid in establishing bona fide pluripotent stem cells in pig.

To have a better understanding of pluripotency, whole gene expression of embryo-derived stem cells (EdSCs) in bovine species was investigated. EdSCs were established from the embryos produced by in vitro fertilization, parthenogenesis and somatic cell nuclear transfer. Then, the microarray was performed and analyzed. Differently expressed genes (DEGs) were also confirmed by Real-time PCR. Among 10,203 DEGs, little difference was found in gene expression among three kinds of EdSCs. Conversely, all EdSCs have an immensely different gene expression when compared with somatic cells, consistent with scatter plat results. To investigate shared pathways for pluripotency in all EdSCs, 2,415 co-DEGs were identified which compared with somatic cells. By KEGG database, there were 54 signaling pathways in co-DEGs and some of them were related with pluripotency maintenance such as TGFβ, WNT and JAK-STAT signaling. In TGFβ signaling, BMP family and SMAD family were involved in co-up-regulated DEGs. In WNT signaling, WNT family and receptors were included in co-up-regulated DEGs, while inhibitors of WNT signaling were associated with co-down-regulated DEGs. In JAK-STAT signaling, STAT3 belonged to co-down-regulated DEGs. These DEGs were also confirmed by Real-time PCR. Taken together, BMP and WNT pathways may be activated and paly central roles to retain pluripotency in bovine EdSCs, whereas the LIF/STAT3 pathway may not be operated well. This study was supported by a grant from the National Research Foundation of Korea (NRF-2006-2004042, and No. 2015048003 through the Oromaxillofacial Dysfunction Research Center for the Elderly at Seoul National University) and the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture, Food and Rural Affairs (MAFRA; 111160-04), Republic of Korea.

The present study was conducted to investigate the effect of caffeine and theophilline on sperm motility during in vitro fertilization (IVF). Briefly, commercial boar semen was centrifuged and resuspended (5x107 sperms/ml) with fertilization medium (mTBM) supplemented with 2 mM caffeine (Caf), 5 mM theophylline (The), 2 mM caffeine + 5mM theophylline (Caf + The), and not supplemented control. The semen parameters of the four groups were analyzed by computer-assisted semen analysis (CASA, Medical Supply Co. Ltd) system at 6 h as time for IVF at 38.5 C under 5 % CO2 in air. The groups were examined 11 semen parameters such as total motility (TM), curvilinier velocity (VCL), straight-line velocity (VSL), average-path velocity (VAP), and hyperactivated (HYP), etc. A total motility of control group (41.3 %) at 6 h showed significantly (P<0.05) higher than those of other groups (Caf, 37.2; The, 35.2; Caf + The, 30.1 %). Results from many other sperm parameters indicated that the theophylline and caffeine negatively affected on sperm motility during IVF. These results suggested that the supplementation of caffeine and/or theophylline was not essential for IVF in pigs. To prove this suggestion, further studies are needed to analyze fertility and embryonic development after IVF with or without the supplementation of caffeine and/or theophylline.