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.

This study was performed to comprehend the plasma proteins expressed specifically during early pregnancy in pregnant or non-pregnant Hanwoo using proteomic analysis technique. Plasma samples (0, 2, 3, 4, 7, and 11 weeks after AI) were obtained from pregnant (P, n=3) or non-pregnant (NP, n=4) Hanwoo, respectively. To evaluate proteins differentially expressed, 2-dimensional electrophoresis (2DE) was conducted. Normalized protein spots were selected for the significant expression variation deviated over two fold in its expression level between two groups. Molecular functions of the proteins were DNA binding, protein binding, hemoglobin binding, ferrochelatase and transporter activity and arylestera, respectively. According to western blotting, haptoglobin was specifically expressed only in NP group during early pregnancy; however, paraoxonase 1 was highly expressed in pregnant group. Based on these results, pregnancy was maintained successfully by the activation of specific plasma proteins associated with immune system and antioxidant regulation during early pregnancy in Hanwoo