DIRECT GENE REPLACEMENT OF THE MOUSE ALPHA(1,3)-GALACTOSYLTRANSFERASEGENE WITH HUMAN ALPHA(1,2)-FUCOSYL-TRANSFERASE GENE - CONVERTING ALPHA-GALACTOSYL EPITOPES INTO H-ANTIGENS

The chronic donor organ shortage has led to the production of transgen ic animals, We assume that cells or organs derived from possible anima l donors carrying a large amount of alpha-galactosyl epitopes should n ot be transplanted into humans, because a corresponding amount of immu nosuppressants would be needed to prolong the survival of such xenogra fts in the recipients. This may not only make the recipients compromis ed hosts but also introduce some unknown or uncontrollable pathogens i nto society at large. We also assume that gene manipulation itself sho uld not be a detriment to possible transgenic animals. To explore poss ibilities that not only can minimize the possible detrimental factors to humans, such as alpha-galactosyl epitopes, but also can minimize th e possible detriment to transgenic animals, such as random integration of the extraneous genes with or without uncontrollable regulatory seq uences, we have produced a DNA construct that replaces the mouse alpha (1,3)-galactosyltransferase gene (GT) with the human alpha(1,2)-fucosy ltransferase (FT) minigene (promoterless for the expression of FT) at the GT locus. The mouse fibrosarcoma cell line, L929, was transfected with the construct. Colonies were obtained after incubation with non-h eat-inactivated human serum. Southern blot analysis demonstrated that one allele of the mouse GT gene was replaced with the FT minigene at t he GT locus without integration of any selectable marker genes. The im munostaining analysis with lectins showed that the transfectants expre ssed H antigens, which suggested that H antigens were expressed by the intrinsic GT promoter. Thus gene replacement, knock-in, of the mouse GT with the human FT without integration of any selectable marker gene s in the GT locus was shown to be possible. This is especially importa nt in producing transgenic animals for the clinical application of xen ografts into humans.