title: Transgenic pigs expressing human decay-accelerating factor regulated by porcine MCP gene promoter.
abstract: Porcine membrane cofactor protein (pMCP) is abundantly expressed throughout the body with particularly strong expression on the vascular endothelia. Previous studies demonstrated that the promoter of the pMCP gene induced efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55), in transgenic mice. In the present study, we tried to produce transgenic pigs with two hybrid genes, 0.9/hDAF and 5.4/hDAF, which were composed of human DAF (hDAF) gene regulated under pMCP promoters of different lengths (0.9 and 5.4 kb). Five live founder transgenic pigs were obtained only with the 0.9/hDAF construct. Although, four founder pigs transmitted the transgene to the second generation, the transmission rates varied among founders. We examined the expression of hDAF in tissues of descendants of two lines (Dm1 and Dm4). Human DAF specific RNAs were confirmed by an RT-PCR analysis in all organs examined. Levels of hDAF protein in the organs from the descendants of Dm1 line were higher than those in the corresponding human organs as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies showed that the tissue distribution of hDAF in the descendants of both lines was similar to that of endogenous pMCP. The expression level of hDAF on the vascular endothelial cells in Dm1 line was twice that on the corresponding human cells. We tested whether proinflammatory cytokines upregulate an efficiency of pMCP promoter on hDAF expression in transgenic pigs. Although the expression of hDAF on the human endothelial cells increased with a combination of cytokines, tumor necrosis factor alpha and interferon-gamma, no cytokine-induced upregulation was seen in the cells of transgenic pigs. The endothelial cells from transgenic pigs exhibited high resistance to the human serum-mediated cytolysis.
authors.affiliation: The Animal Engineering Research Institute, Tsukuba, Ibaraki, Japan. murakami@rdc.nipponham.co.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Murakami
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagashima
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Takahagi
authors.forename: Shuji
authors.initials: S
authors.lastname: Miyagawa
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Fujimura
authors.forename: Koji
authors.initials: K
authors.lastname: Toyomura
authors.forename: Rie
authors.initials: R
authors.lastname: Nakai
authors.forename: Mako
authors.initials: M
authors.lastname: Yamada
authors.forename: Takashi
authors.initials: T
authors.lastname: Kurihara
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Shigehisa
authors.forename: Masaru
authors.initials: M
authors.lastname: Okabe
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Seya
authors.forename: Ryota
authors.initials: R
authors.lastname: Shirakura
authors.forename: Taroh
authors.initials: T
authors.lastname: Kinoshita
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015703:Antigens, CD / Q000235:genetics*; D015153:Blotting, Western; D018958:CD55 Antigens / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000235:genetics*; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine / Q000235:genetics*; D019076:Transgenes / Q000235:genetics
chemical_list: D015703:Antigens, CD; C497857:CD46 protein, human; D018958:CD55 Antigens; D016207:Cytokines; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D012333:RNA, Messenger