Direct gene replacement of the mouse alpha(1,3)-galactosyltransferase gene with human alpha(1,2)-fucosyltransferase gene: Converting alpha-galactosyl epitopes into H antigens

The chronic donor organ shortage has led to the production of transgenic animals, We assume that cells or organs derived from possible animal donors carrying a large amount of alpha-galactosyl epitopes should not be transplanted into humans, because a corresponding amount of immunosuppressants would be needed to prolong the survival of such xenografts in the recipients. This may not only make the recipients compromised hosts but also introduce some unknown or uncontrollable pathogens into society at large. We also assume that gene manipulation itself should not be a detriment to possible transgenic animals. To explore possibilities that not only can minimize the possible detrimental factors to humans, such as alpha-galactosyl epitopes, but also can minimize the possible detriment to transgenic animals, such as random integration of the extraneous genes with or without uncontrollable regulatory sequences, we have produced a DNA construct that replaces the mouse alpha(1,3)-galactosyltransferase gene (GT) with the human alpha(1,2)-fucosyltransferase (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-heat-inactivated human serum. Southern blot analysis demonstrated that one allele of the mouse GT gene was replaced with the FT minigene at the GT locus without integration of any selectable marker genes. The immunostaining analysis with lectins showed that the transfectants expressed 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 genes in the GT locus was shown to be possible. This is especially important in producing transgenic animals for the clinical application of xenografts into humans.