Down-regulation of αGal epitopes by co-transfection of α1,3-galactosidase gene and α1,2-fucosyltransferase gene

Abstract

The polycarbohydrate structure of Galα1-3Galβ1-4GluNAc-R (known as αGal epitopes of xenoantigen), produced by α1-3-galactosyltransferase (α1,3-GT) in the course of animal development, is the major xenoantigen on the cell surface of porcine which causes hyperacute rejection in pig-to-human xenotransplantation. Alpha-1,3-galactosidase (AGL), a hydrolytic enzyme, can remove the terminal α-1,3-galactosyl from the Galα1-3Galβ1-4GluNAc-R structure resulting in cleaning αGal epitopes from the porcine cells. Alpha-1,2-fucosyltransferase (HT) can modify the surface carbohydrate phenotype of porcine cells, bringing about reduction of αGal epitopes expression. In this study, human AGL and HT gene were co-transfected to porcine fetal fibro-blast (PFFb) in equimolar concentration to reduce the xenoantigen. Gene and protein of hAGL and HT were both detected to express at high level by RT-PCR and Western blot, respectively. There was an 84% reduction in αGal xenoantigen and an 82% increase in H antigen as assayed by flow cytometry in the AGL and HT gene co-transfected PFFb. The number and morphology of transgenic PFFb chromosome were normal. Findings indicate that Galα1-3Gal epitopes of PFFb could be down regulated byAGL and HT co-transfection without deleterious effects on the chromosomal profile of the transgenic cell.