C. Koike

Introduction of α(1,2)‐fucosyltransferase and its effect on a‐Gal epitopes in transgenic pig

Abstract: Hyperacute rejection (from pig to human) is thought to result from activation of complement initiated by the binding of host natural antibodies to α‐galactosyl (α‐Gal) epitopes of donor endothelial cells. However, α‐Gal epitope shares a common precursor with H antigen in humans. This means that H antigens as well as α‐Gal epitopes are synthesized in a competitive manner by different enzymes. We thought that it would be possible to convert α‐Gal epitopes into H antigens by introducing cDNA of α(1,2)‐fucosyltransferase (α1–2FT) into porcine cells, and so, pig embryos were microinjected with αl‐2FT cDNA. Transgenic pigs that carried α1–2FT were thus established. Cytotoxicity of fibrocytes derived from skin of transgenic pig was measured by 51Cr release assay, which showed that H antigen‐expressing cells were significantly resistant to a challenge with human sera. These experiments indicate that our method provides a new strategy which contributes to a successful discordant xenotransplantation.

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

Abstract: 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 α‐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 α‐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 oc(1,3)‐galactosyltransferase gene (GT) with the human a(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.

Establishment of a human DAF/HRF20 double transgenic mouse line is not sufficient to suppress hyperacute rejection

To solve the chronic donor organ shortage, the pig is considered to be a possible donor candidate for human transplantation. However, hyperacute rejection occurs due to the activation of the complement cascade. Therefore, the introduction of human complement inhibitors into animal cells has been proposed as a means to prevent such exologous complement activation. To investigate the extent to which complement inhibitors are resistant to human sera in discordant animals, we established transgenic mice lines which expressed either human decay-accelerating factor (DAF) and/or homologous restriction factor 20 (HRF20) using microinjection methods. Human sera were injected into (a) 10 control mice, (b) 10 DAF-transgenic mice, (c) 10 HRF20-transgenic mice, and (d) 10 DAF and HRF20-transgenic mice. The results showed that all the mice in groups a, b, and c died immediately after injection. Three of the mice in group d died, while seven survived but showed hyperpnea and low activity. The pathological findings of groups a, b, and c included severe coagulation; however, the survivors of group d showed less severe symptoms. The above findings thus suggest that both DAF and HRF20 tend to prevent complement activation to some extent; however, its effectiveness is not considered to be sufficient for clinical use in transplantation.