20 SIALYLTRANSFERASE GENE EXPRESSION IN GGTA1 KNOCKOUT PIGS

Abstract

It is considered that GGTA1 knockout (KO) pig production via somatic cell NT would overcome the problem of immune rejection after xenotransplantation. It is reported that although GGTA KO mice showed only a mild increase in sialyltransferase gene expression, GGTA1 deficiency in pig could increase the sialyltransferase activities, non-Gal epitope expression, consequently may raise non-Gal xenoantigenicity. Therefore, in the present study we investigated whether the expression level of Sia-containing glycoconjugate mRNA in transgenic pigs could be affected by knocking out the GGTA1 gene. Besides GGTA1 KO pigs, double genes overexpressing pigs (2TG) and GGTA1 KO with double genes overexpressing (KO+2TG) pigs were produced by somatic cell NT. For the present study, fibroblasts were isolated from wild-type pigs without gene modification, 2TG, GGTA1 KO, and KO+2TG pig. The GAPDH gene was used as an internal standard to normalise the real-time PCR (RT-qPCR) analysis reaction efficiency and to quantify mRNA in pigs-derived fibroblast. The expression levels were compared between them (RT-qPCR) in triplicate for each sample. Oligonucleotide primers for real-time PCR were designed for Hanganutziu-Deicher antigen (ST3Gal1–4, ST6Gal1) and Sialyl-Tn antigen (ST6GalNac1, ST6GalNac2, and ST6GalNac6) analysis. For statistical analysis, one-way ANOVA with Dunn’s multiple comparison test were used. The mRNA expression of GGTA1 KO and KO+2TG pig derived fibroblasts cells genes showed that ST3Gal1, ST3Gal2, ST3Gal3, and ST6Gal1 gene expression were significantly up-regulated compared to the wild and 2TG pigs (P < 0.05). However, ST3Gal4, Sialyl-Tn antigen including ST6GalNac1, ST6GalNac2, and ST6GalNac6 in KO+2TG pigs were not different compared with the wild pigs (P > 0.05), whereas only GGTA1 KO pigs showed significantly higher expressions than wild, 2TG, and KO+2TG pigs (P < 0.05). These results demonstrated that GGTA KO pig-derived cells exhibit a higher Hanganutziu-Deicher antigen on glycoprotein and glycolipid than controls, and KO+2TG pig exhibit no differences when compared with GGTA1 KO pig, indicating that they do not act as an immune antigen in xenograft. Overall, the increase in glycosyltransferase expression suggests a corresponding increase in the cell surface sialyation in GGTA KO pig cells. For xenotransplantation, KO+2TG pigs were more preferable because of absence of immune rejection for Sia-containing glycoconjugate on glycoprotein and glycolipid than GGTA KO pigs.

This study was supported by the Ministry of Trade, Industry and Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.