Abstract. Galα1-3Gal (α-Gal epitope) is the major xenoantigenic epitope responsible for hyperacute rejection upon pigto-human xenotransplantation. Endo-β-galactosidase C (EndoGalC) from Clostridium perfringens can digest the α-Gal epitope. In this study, gene-engineered primary cultured porcine embryonic fibroblasts (PEF) expressing EndoGalC were obtained and subjected to somatic cell nuclear transfer (SCNT) to test whether xenograft-competent pigs can be created. The EndoGalC-expressing PEF clones exhibited highly reduced expression of α-Gal epitope, as revealed by cytochemical staining with BS-I-B4 isolectin, a lectin that specifically binds to α-Gal epitope, and FACS analysis. The pattern of low level of α-Gal epitope expression continued for at least 6 months (more than 10 generations) after isolation. SCNT of nuclei from these cells resulted in the generation of blastocysts that displayed nearly complete loss of α-Gal epitope from their cell surface. This is the first study to demonstrate that SCNT using EndoGalC-expressing PEFs as donors would be useful for production of genetically modified cloned pigs suitable for xenotransplantation. Key words: Endo-β-galactosidase C, Gene-modified, Miniature pig, Somatic cell nuclear transfer, Xenotransplantation (J. Reprod. Dev. 56: [630][631][632][633][634][635][636][637][638] 2010) ig-to-human xenotransplantation leads to an immediate rejection process, termed hyperacute rejection (HAR), caused by the binding of natural antibodies present in humans to the carbohydrate epitope gal α-1,3-gal (α-Gal epitope) that is present in pigs but not in humans [1][2][3]. This epitope is synthesized mainly by α-1,3-galactosyltransferase (α-1,3-GalT-1), and removal of the epitope is considered a prerequisite for xenotransplantation [4].Several approaches have been proposed to eliminate the α-Gal epitope from pigs. Removal of the α-1,3-GalT-1 gene by gene targeting is a straightforward method. In fact, in pigs, successful gene targeting has been achieved by a somatic cell nuclear transfer (SCNT) technique using targeted fibroblast clones [5][6][7][8][9][10]. Another approach is employment of endo-β-galactosidase C (EndoGalC). This enzyme is derived from Clostridium perfringens and can digest the α-Gal epitope by cleaving the α-galactosidic linkage [11]. EndoGalC is thus considered an effective means of enzymatic removal of the α-Gal epitope from the surface of animal cells such as porcine cells [12][13][14]. However, no attempt to produce genetically engineered pigs expressing EndoGalC systemically has yet been made.Several approaches have been considered for the production of pigs overexpressing the EndoGalC gene. [19][20][21][22]. Of these approaches, the SCNT-mediated transgenesis approach appears to be most promising, since donor cells highly expressing a transgene can easily be selected prior to SCNT. In this case, the most important point requiring consideration is how EndoGalC is ubiquitously expressed through each organ of the SCNT pig. We therefore employed two types of...
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