To avoid hyperacute rejection of xenografts, α1,3-galactosyltransferase knock-out (GalT KO) pigs have been produced. In this study, we examined whether Sia-containing glycoconjugates are important as an immunogenic non-Gal epitope in the pig liver with disruption of α1,3-galactosyltransferase gene. The target cells were then used as donor cells for somatic cell nuclear transfer (scNT). A total of 1,800 scNT embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. Real-time RT-PCR and glycosyltransferase activity showed that α2,3-sialyltransferase (α2,3ST) and α2,6-sialyltransferase (α2,6ST) in the heterozygote GalT KO liver have higher expression levels and activities compared to controls, respectively. According to lectin blotting, sialic acidcontaining glycoconjugate epitopes were also increased due to the decreasing of α-Gal in heterozygote GalT KO liver, whereas GalNAc-containing glycoconjugate epitopes were decreased in heterozygote GalT KO liver compare to the control. Furthermore, the heterozygote GalT KO liver showed a higher Neu5Gc content than control. Taken together, these finding suggested that the deficiency of GalT gene in pigs resulted in increased production of Neu5Gc-bounded epitopes (H-D antigen) due to increase of α2,6-sialyltransferase. Thus, this finding suggested that the deletion of CMAH gene to the GalT KO background is expected to further prolong xenograft survival.

ABSTRACT   INTRODUCTION   MATERIALS AND METHODS    Construction of α1,3-galactosyltransferase Gene KO Vector    PCR Analysis of Targeted Cells    Production of Heterozygote GalT KO Pigs    Sample Preparation and Protein Determination    RNA Isolation and Real-time RT-PCR    Assay of Glycosyltransferase Activity    Lectin Blotting    Analysis of Sialic Acid Contents    Statistical Analysis   RESULTS    Production of Heterozygote GalT KO Gnotobiotic Miniature Pigs    Heterozygote GalT KO Pig-Derived Liver Displays Higher Non-Gal Xenoantigen   DISCUSSION   REFERENCES

• Gyesik Min(Department of Pharmaceutical Engineering, Gyeongnam National University of Science & Technology)
• Jong-Yi Park(Department of Animal Biotechnology, Konkuk University) Corresponding author