title: Generation of α-1,3-galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes.
abstract: Recent progress in genetic manipulation of pigs designated for xenotransplantation ha6s shown considerable promise on xenograft survival in primates. However, genetic modification of multiple genes in donor pigs by knock-out and knock-in technologies, aiming to enhance immunological tolerance against transplanted organs in the recipients, has not been evaluated for health issues of donor pigs. We produced transgenic Massachusetts General Hospital piglets by knocking-out the α-1,3-galactosyltransferase (GT) gene and by simultaneously knocking-in an expression cassette containing five different human genes including, DAF, CD39, TFPI, C1 inhibitor (C1-INH), and TNFAIP3 (A20) [GT-(DAF/CD39/TFPI/C1-INH/TNFAIP3)/+] that are connected by 2A peptide cleavage sequences to release individual proteins from a single translational product. All five individual protein products were successfully produced as determined by western blotting of umbilical cords from the newborn transgenic pigs. Although gross observation and histological examination revealed no significant pathological abnormality in transgenic piglets, hematological examination found that the transgenic piglets had abnormally low numbers of platelets and WBCs, including neutrophils, eosinophils, basophils, and lymphocytes. However, transgenic piglets had similar numbers of RBC and values of parameters related to RBC compared to the control littermate piglets. These data suggest that transgenic expression of those human genes in pigs impaired hematopoiesis except for erythropoiesis. In conclusion, our data suggest that transgenic expression of up to five different genes can be efficiently achieved and provide the basis for determining optimal dosages of transgene expression and combinations of the transgenes to warrant production of transgenic donor pigs without health issues.
authors.affiliation: National Institute of Animal Science, Rural Development Administration, Wanju, Jeollabuk-do, 55365, Republic of Korea.
authors.forename: Dae-Jin
authors.initials: DJ
authors.lastname: Kwon
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, Republic of Korea.|Department of Functional Genomics, University of Science and Technology, Daejeon, 34113, Republic of Korea.
authors.forename: Dong-Hwan
authors.initials: DH
authors.lastname: Kim
authors.affiliation: National Institute of Animal Science, Rural Development Administration, Wanju, Jeollabuk-do, 55365, Republic of Korea.
authors.forename: In-Sul
authors.initials: IS
authors.lastname: Hwang
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, Republic of Korea.
authors.forename: Dong-Ern
authors.initials: DE
authors.lastname: Kim
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, Republic of Korea.
authors.forename: Hyung-Joo
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, Republic of Korea.
authors.forename: Jang-Seong
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Department of Animal Sciences, The Ohio State University, Columbus, OH, 43210, USA.
authors.forename: Kichoon
authors.initials: K
authors.lastname: Lee
authors.affiliation: National Institute of Animal Science, Rural Development Administration, Wanju, Jeollabuk-do, 55365, Republic of Korea.
authors.forename: Gi-Sun
authors.initials: GS
authors.lastname: Im
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, Republic of Korea. jwlee@kribb.re.kr.|Department of Functional Genomics, University of Science and Technology, Daejeon, 34113, Republic of Korea. jwlee@kribb.re.kr.
authors.forename: Jeong-Woong
authors.initials: JW
authors.lastname: Lee
authors.affiliation: National Institute of Animal Science, Rural Development Administration, Wanju, Jeollabuk-do, 55365, Republic of Korea. hwangss@korea.kr.
authors.forename: Seongsoo
authors.initials: S
authors.lastname: Hwang
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics* / Q000254:growth & development; D015703:Antigens, CD / Q000235:genetics; D001081:Apyrase / Q000235:genetics; D050718:Complement C1 Inhibitor Protein / Q000235:genetics; D004920:Erythropoiesis / Q000235:genetics*; D005700:Galactosyltransferases / Q000235:genetics*; D005786:Gene Expression Regulation; D055786:Gene Knockout Techniques; D006410:Hematopoiesis / Q000235:genetics; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics; D013552:Swine; D019076:Transgenes / Q000235:genetics*; D014183:Transplantation, Heterologous; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3 / Q000235:genetics
chemical_list: D015703:Antigens, CD; D050718:Complement C1 Inhibitor Protein; D008074:Lipoproteins; C051928:lipoprotein-associated coagulation inhibitor; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D001081:Apyrase; C067869:CD39 antigen
keywords: Immune rejection; Leucopenia; Multi-transgenic pigs; Thrombocytopenia; Xenotransplantation