title: Successful cross-breeding of cloned pigs expressing endo-beta-galactosidase C and human decay accelerating factor.
abstract: BACKGROUND
  For successful organ xenotransplantation, genetically engineered pigs have been actively produced. Our attention has focused on (i) reduction of alphaGal expression by its digestion enzyme, endo-beta-galactosidase C (EndoGalC), and (ii) inhibition of complement activation by human decay accelerating factor (hDAF). Cell sorting and nuclear transfer enabled the effective production of cloned pigs expressing transgene at high levels. We report the successful cross-breeding of pigs expressing EndoGalC and hDAF.
  
  
  METHODS
  After hDAF and EndoGalC genes were transfected into pig fibroblasts from the fetus of Landrace x Yorkshire and Meishan, respectively, transfected cells expressing transgenes effectively were collected using a cell sorter. Cloned pigs were produced using the technology of somatic cell nuclear transfer. After cross-breeding of cloned pigs, kidneys expressing both EndoGalC and hDAF were transplanted into baboons to examine the efficacy of gene transduction.
  
  
  RESULTS
  Well-designed cloned pigs were produced by cross-breeding. alphaGal expression levels in cloned pigs were reduced up to 2 to 14%, compared to that in wild-type pigs. hDAF expression reached about 10- to 70-fold, compared to that in human umbilical vein endothelial cells. No congenital deformity was observed. There was no problem of increased stillbirth rate or growth retardation. Hyperacute rejection could be avoided in such a cloned pig to baboon kidney transplantation without any treatment for anti-pig antibody removal. However, grafts suffered from fibrin deposition as early as 1 h after transplantation, and were rejected after 1 week.
  
  
  CONCLUSIONS
  Using a cell sorting system for effective collection of transfected cells, two types of cloned pigs were produced with a very high level of hDAF expression and a low level of alphaGal expression. Such genetic modification was effective in preventing hyperacute rejection, but there was an immediate lapse into procoagulation after transplantation, resulting in acute vascular rejection. Effective suppression of antibody binding to the graft would be necessary, even if a high level of hDAF is expressed.
authors.affiliation: Prime Tech Ltd, Tsuchiura, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Yazaki
authors.forename: Masaki
authors.initials: M
authors.lastname: Iwamoto
authors.forename: Akira
authors.initials: A
authors.lastname: Onishi
authors.forename: Yuko
authors.initials: Y
authors.lastname: Miwa
authors.forename: Shunichi
authors.initials: S
authors.lastname: Suzuki
authors.forename: Dai-ichiro
authors.initials: D
authors.lastname: Fuchimoto
authors.forename: Shoichiro
authors.initials: S
authors.lastname: Sembon
authors.forename: Tadashi
authors.initials: T
authors.lastname: Furusawa
authors.forename: Michiko
authors.initials: M
authors.lastname: Hashimoto
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Oishi
authors.forename: DaGe
authors.initials: D
authors.lastname: Liu
authors.forename: Takaharu
authors.initials: T
authors.lastname: Nagasaka
authors.forename: Takafumi
authors.initials: T
authors.lastname: Kuzuya
authors.forename: Shoichi
authors.initials: S
authors.lastname: Maruyama
authors.forename: Haruko
authors.initials: H
authors.lastname: Ogawa
authors.forename: Kenji
authors.initials: K
authors.lastname: Kadomatsu
authors.forename: Kazuharu
authors.initials: K
authors.lastname: Uchida
authors.forename: Akimasa
authors.initials: A
authors.lastname: Nakao
authors.forename: Takaaki
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D018958:CD55 Antigens / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D019976:Cloning, Organism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006824:Hybridization, Genetic; D008297:Male; D053652:Nuclear Transfer Techniques; D010215:Papio; D010375:Pedigree; D034421:Sus scrofa; D019076:Transgenes; D014183:Transplantation, Heterologous; D000519:alpha-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D018958:CD55 Antigens; D006026:Glycoside Hydrolases; C057030:endo-beta-D-galactosidase C; D000519:alpha-Galactosidase