title: Pigs transgenic for human thrombomodulin have elevated production of activated protein C.
abstract: BACKGROUND
  The inability of porcine thrombomodulin (TM) to activate human anticoagulant protein C after pig-to-human xenotransplantation may lead to an aberrant activation of coagulation with microthrombosis and ultimately failure of the transplanted organ. Here, we describe the production of triple-transgenic pigs expressing hCD59/DAF and human thrombomodulin (hTM) and tested hTM-transgenic fibroblasts obtained from these pigs for their ability to activate human protein C in a new in vitro assay.
  
  
  METHODS
  Fibroblast cell cultures were established from a hCD59/DAF transgenic pig and transfected with a vector coding for hTM under transcriptional control of the CMV promoter. Transfected cells were analyzed for integration and expression of the hTM vector by PCR and RT-PCR. One cell clone was used as donor for somatic cell nuclear transfer to produce triple transgenic (CD59/DAF/hTM) pigs. Pigs were characterized in detail with regard to hTM integration and expression by PCR, RT-PCR, Northern blot, Western blot, immunostaining, and FACS analysis. Fibroblasts from hTM-transgenic pigs were analyzed in a new in vitro hTM coactivity assay to assess the production of activated protein C (aPC) and results were compared to those from wild-type controls.
  
  
  RESULTS
  In total, 1040 cloned transgenic embryos were transferred to eight recipients. Five recipients remained pregnant and delivered 22 piglets. Expression of hTM was detected in all xenorelavant organs including heart, liver, kidney, lung, and pancreas. The lowest levels of expression were found in lung and liver while all animals showed a strong, but frequently patchy expression pattern of hTM in heart, kidney, and pancreas. The hTM cofactor activity (ranging on a scale from 5-18 U/10(5) cells/2h) was significantly higher in fibroblasts of hTM-transgenic clones compared to wild-type porcine fibroblasts (1.7 U/10(5) cells/2h).
  
  
  CONCLUSIONS
  For the first time, healthy hTM-transgenic pigs could be successfully generated by somatic cell nuclear transfer. hTM can be expressed in porcine organs without perturbation of the porcine coagulation system. hTM-transgenic porcine fibroblasts showed elevated aPC production in an in vitro hTM coactivity assay. These findings warrant further work on the control of the xenogenic activation of coagulation by transgenic approaches.
authors.affiliation: Institute of Farm Animal Genetics, Department of Biotechnology, Mariensee, Neustadt, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Petersen
authors.forename: Wolf
authors.initials: W
authors.lastname: Ramackers
authors.forename: Andreas
authors.initials: A
authors.lastname: Tiede
authors.forename: Andrea
authors.initials: A
authors.lastname: Lucas-Hahn
authors.forename: Doris
authors.initials: D
authors.lastname: Herrmann
authors.forename: Brigitte
authors.initials: B
authors.lastname: Barg-Kues
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schuettler
authors.forename: Lars
authors.initials: L
authors.lastname: Friedrich
authors.forename: Reinhard
authors.initials: R
authors.lastname: Schwinzer
authors.forename: Michael
authors.initials: M
authors.lastname: Winkler
authors.forename: Heiner
authors.initials: H
authors.lastname: Niemann
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D018957:CD59 Antigens / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008297:Male; D053652:Nuclear Transfer Techniques; D011247:Pregnancy; D011486:Protein C / Q000235:genetics / Q000378:metabolism*; D013552:Swine / Q000235:genetics*; D018180:Thrombomodulin / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution; D014183:Transplantation, Heterologous
chemical_list: D018957:CD59 Antigens; D011486:Protein C; D018180:Thrombomodulin