title: Kidneys From α1,3-Galactosyltransferase Knockout/Human Heme Oxygenase-1/Human A20 Transgenic Pigs Are Protected From Rejection During Ex Vivo Perfusion With Human Blood.
abstract: Multiple modifications of the porcine genome are required to prevent rejection after pig-to-primate xenotransplantation. Here, we produced pigs with a knockout of the α1,3-galactosyltransferase gene (GGTA1-KO) combined with transgenic expression of the human anti-apoptotic/anti-inflammatory molecules heme oxygenase-1 and A20, and investigated their xenoprotective properties.
  
  
  METHODS
  The GGTA1-KO/human heme oxygenase-1 (hHO-1)/human A20 (hA20) transgenic pigs were produced in a stepwise approach using zinc finger nuclease vectors targeting the GGTA1 gene and a Sleeping Beauty vector coding for hA20. Two piglets were analyzed by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and sequencing. The biological function of the genetic modifications was tested in a (51)Chromium release assay and by ex vivo kidney perfusions with human blood.
  
  
  RESULTS
  Disruption of the GGTA1 gene by deletion of few basepairs was demonstrated in GGTA1-KO/hHO-1/hA20 transgenic pigs. The hHO-1 and hA20 mRNA expression was confirmed by quantitative reverse-transcription polymerase chain reaction. Ex vivo perfusion of 2 transgenic kidneys was feasible for the maximum experimental time of 240 minutes without symptoms of rejection.
  
  
  CONCLUSIONS
  Results indicate that GGTA1-KO/hHO-1/hA20 transgenic pigs are a promising model to alleviate rejection and ischemia-reperfusion damage in porcine xenografts and could serve as a background for further genetic modifications toward the production of a donor pig that is clinically relevant for xenotransplantation.
authors: Institute of Farm Animal Genetics, Friedrich-Loeffler Institut, Mariensee, Neustadt, Germany.
authors: Hellen E
authors: HE
authors: Ahrens
authors: Institute of Farm Animal Genetics, Friedrich-Loeffler Institut, Mariensee, Neustadt, Germany.
authors: Björn
authors: B
authors: Petersen
authors: Transplant Laboratory, Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors: Wolf
authors: W
authors: Ramackers
authors: Institute of Farm Animal Genetics, Friedrich-Loeffler Institut, Mariensee, Neustadt, Germany.
authors: Stoyan
authors: S
authors: Petkov
authors: Institute of Farm Animal Genetics, Friedrich-Loeffler Institut, Mariensee, Neustadt, Germany.
authors: Doris
authors: D
authors: Herrmann
authors: Institute of Farm Animal Genetics, Friedrich-Loeffler Institut, Mariensee, Neustadt, Germany.
authors: Janet
authors: J
authors: Hauschild-Quintern
authors: Institute of Farm Animal Genetics, Friedrich-Loeffler Institut, Mariensee, Neustadt, Germany.
authors: Andrea
authors: A
authors: Lucas-Hahn
authors: Institute of Farm Animal Genetics, Friedrich-Loeffler Institut, Mariensee, Neustadt, Germany.
authors: Petra
authors: P
authors: Hassel
authors: Institute of Farm Animal Genetics, Friedrich-Loeffler Institut, Mariensee, Neustadt, Germany.
authors: Maren
authors: M
authors: Ziegler
authors: Transplant Laboratory, Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors: Wiebke
authors: W
authors: Baars
authors: Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors: Sabine
authors: S
authors: Bergmann
authors: Transplant Laboratory, Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors: Reinhard
authors: R
authors: Schwinzer
authors: Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
authors: Michael
authors: M
authors: Winkler
authors: Institute of Farm Animal Genetics, Friedrich-Loeffler Institut, Mariensee, Neustadt, Germany.
authors: Heiner
authors: H
authors: Niemann