Human HLA-Ev (147) Expression in Transgenic Animals.
BACKGROUND
In our previous study, we reported on the development of substituting S147C for HLA-E as a useful gene tool for xenotransplantation. In this study we exchanged the codon of HLA-Ev (147), checked its function, and established a line of transgenic mice.


METHODS
A new construct, a codon exchanging human HLA-Ev (147) + IRES + human beta 2-microgloblin, was established. The construct was subcloned into pCXN2 (the chick beta-actin promoter and cytomegalovirus enhancer) vector. Natural killer cell- and macrophage-mediated cytotoxicities were performed using the established the pig endothelial cell (PEC) line with the new gene. Transgenic mice with it were next produced using a micro-injection method.


RESULTS
The expression of the molecule on PECs was confirmed by the transfection of the plasmid. The established molecules on PECs functioned well in regulating natural killer cell-mediated cytotoxicity and macrophage-mediated cytotoxicity. We have also successfully generated several lines of transgenic mice with this plasmid. The expression of HLA-Ev (147) in each mouse organ was confirmed by assessing the mRNA. The chick beta-actin promoter and cytomegalovirus enhancer resulted in a relatively broad expression of the gene in each organ, and a strong expression in the cases of the heart and lung.


CONCLUSIONS
A synthetic HLA-Ev (147) gene with a codon usage optimized to a mammalian system represents a critical factor in the development of transgenic animals for xenotransplantation.
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan. Electronic address: matsuura@pedsurg.med.osaka-u.ac.jp.
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Matsuura
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Maeda
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Sakai
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Eguchi
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Lo
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Hasuwa
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Ikawa
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Nakahata
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Zenitani
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Yamamichi
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Umeda
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Deguchi
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Okuyama
Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
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Miyagawa
D000199:Actins / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D003062:Codon / Q000235:genetics*; D003587:Cytomegalovirus; D042783:Endothelial Cells / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005813:Genes, Synthetic; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D007694:Killer Cells, Natural / Q000502:physiology; D008264:Macrophages / Q000502:physiology; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D013552:Swine; D014162:Transfection; D014183:Transplantation, Heterologous; D000097625:HLA-E Antigens
D000199:Actins; D003062:Codon; D015395:Histocompatibility Antigens Class I; D000097625:HLA-E Antigens