Medycyna Wet. 66 (5), 316-322, 2010
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| Lipiński D., Jura J., Zeyland J., Juzwa W., Mały E., Kalak R., Bochenek M., Plawski A., Szalata M., Smorąg Z., Słomski R. |
| Production of transgenic pigs expressing human α1,2-fucosyltransferase to avoid humoral xenograft rejection |
| The use of animals as a source of organs and tissues for xenotransplantation may overcome the growing shortage of human organ donors. However, the presence of xenoreactive antibodies in humans, directed against the swine Gal antigen present on the surface of xenograft donor cells, leads to the complement activation and an immediate xenograft rejection as a consequence of hyperacute reaction. In order to prevent a hyperacute rejection, it is possible to alter the swine genome with human genes modifying the set of the donors cell surface proteins. The aim of this study was to prepare a pCMVFut genetic construct and then introduce it into the swine genome in order to obtain transgenic pigs expressing human á1,2-fucosyltransferase and thereby avoid a humoral xenograft rejection. The pCMVFut gene construct containing the human gene encoding á1,2-fucosyltransferase enzyme under the human cytomegalovirus immediate early promoter was introduced by microinjection into a male pronucleus of a fertilized porcine oocyte. The screening procedure involved isolating genomic DNA from microsections of pigs ears, the amplification of two PCR fragments and the entire sequencing of positive samples. The mapping of the transgene was performed by fluorescence in situ hybridisation (FISH) and transgene expression, while its impact on the reduction of the Gal epitope level on the surface of pig cells was assessed by flow cytometry of primary cultured skin fibroblasts. The influence of the human complement was measured by testing the sensitivity of nontransgenic and transgenic cells to complement-mediated cytotoxicity upon exposure to human serum. As a result of this experiment, the founder male pig was obtained with the transgene mapping to chromosome 14q28. An RT-PCR analysis revealed the expression of the HT gene in different tissues of transgenic pigs. A flow cytometry analysis revealed a reduction in the level of epitop Gal on the cell surface of skin fibroblasts isolated from transgenic pigs. The complement-mediated cytotoxicity assay showed increased viability of transgenic cells in comparison with nontransgenic ones, which confirmed the protective influence of HT expression. In this study we demonstrated that the constitutive transgenic expression of human H-transferase (á1,2-fucosyltransferase) can decrease the amount of Galá1,3Gal (Gal epitope) on the surface of pig cells, which is consistent with the results of other researchers. The expression of á1,2-fucosyltransferase modified the cell surface carbohydrate phenotype of transgenic pig cells, resulting in the expression of the universally tolerated 0 blood group antigen (H antigen) and a subsequent reduction in the expression of Gal epitope, as evaluated by flow cytometry analysis. Apart from the principal data, the flow cytometry analysis revealed no significant differences between the Gal epitope level achieved by CMVFUT heterozygous boar founder TG 1154 and transgene homozygous pig 433 from the F2 generation. The flow cytometry results were confirmed by the cytotoxicity assay. We found no statistical difference in the survival rate between transgenic homozygous and heterozygous cells under the influence of 50% human serum with an active complement system. Both homozygous and heterozygous cells had the same level of lysis protection. |
| Keywords: fucosyltransferase, swine, transgenesis, xenotransplantation |