Swine models of Human Conditions (2010)

92 Hydrogen peroxide (H2O2) is an oxidant with a long half-life, capable of diffusing across cellular membranes and acting as a signaling molecule in vascular endothelial cells. The association between H2O2 and vasodilators such as nitric oxide (NO) is becoming increasingly important to our understanding to the regulation of vascular tone. Previous studies suggest that these vasodilators may play a critical role in disease states such as atherosclerosis and preeclampsia. The objective of the current study is to determine how NO, produced by endothelial nitric oxide synthase (eNOS),is influenced by H2O2 in the swine vasculature. We have developed cloned transgenic Yucatan minipigs carrying a transgene designed to overexpress human catalase (hCat) in an endothelial-specific manner, subsequently metabolizing endogenous H2O2. Yucatan minipig fetal fibroblasts stably transfected with an hCat overexpression construct were isolated into single-cell populations by fluorescence automated cell sorting (FACS) based on fluorescence of a cotransfected eGFP plasmid. Cells sorted into 96-well plates yielded viable colonies in 30% of the wells. Presence of the hCat transgene in fibroblasts was determined by PCR genotyping. Transgenic fibroblasts were used for nuclear transfer into enucleated oocytes by electrofusion. A minimum of 140 cloned embryos were used for embryo transfer into four surrogates. These embryo transfers yielded pregnancies in all four sows. Cloned piglets from three sows near parturition were delivered by cesarean section. A total of six cloned male Yucatan mini piglets from two of the litters were positive for the tie2-catalase transgene as determined by PCR genotyping. These piglets will be used to compare the endothelial response in transgenic versus wild-type pigs. The similarities between swine and human cardiovascular anatomy and physiology suggest that this pig model will provide critical information on the regulation of eNOS by H2O2 and the mechanism sunderlying human vascular health in response to exercise and in specific disease states. This work is funded by a grant from the NIH (R24 RR018276-05) to MHL. IMPAIRED INCRETIN EFFECT IN TRANSGENIC PIGS EXPRESSING A DOMINANT NEGATIVE RECEPTOR FOR GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE IN THE PANCREATIC ISLETS S. Renner, B. Kesler, N. Herbach, D. C. von Waldthausen, R. Wanke, A. Hofmann, A. Pfeifer and E. Wolf Abstract Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted by specific cell types in the intestine and are responsible for the so-called incretin effect, the phenomenon that an oral glucose load elicits a higher insulin response than does an intravenous glucose load. In patients with type 2 diabetes mellitus the overall incretin effect is reduced. This fact is mostly attributed to a lowered insulinotropic effect of GIP, while the effect of GLP-1 is preserved. In order to better understand the consequences of impaired function of GIP, knockout mice lacking a functional GIP receptor (GIPR) as well as transgenic mice expressing a dominant negative GIPR (GIPR) were established. While GIPR mice show only relatively mild changes in glucose homeostasis, GIPR mice display a distinct diabetic phenotype due to disturbed development of the endocrine pancreas (Herbach et al. 2005 Regul. Pept. 125, 103–117). To further clarify the underlying mechanisms, we used a novel, highly efficient gene transfer technology based on lentiviral vectors (Hofmann et al. 2003 EMBO Rep. 4, 1054–1060; Hofmann et al. 2006 Mol. Ther. 13, 59–66) to generate transgenic pigs expressing a GIPR under the control of the rat Ins2 promoter (RIP). RIP-GIPR transgenic pigs develop normally and do not display diabetes mellitus up to at least one year of age. Weekly measured fasting blood glucose levels in transgenic animals did not show a significant difference compared to control pigs. The same was true for monthly determined fructosamine levels. However, RIP-GIPR transgenic pigs exhibited reduced insulin release and higher glucose levels than nontransgenic littermate controls in an oral glucose tolerance test. The area under the curve (AUC) for insulin was 49% smaller (P < 0.01) and the AUC for glucose 26% larger (P < 0.05) in RIP-GIPR transgenic pigs (n = 5) than in their non-transgenic littermate controls (n = 5). These findings demonstrate that expression of a GIPR, which was shown by RT-PCR in isolated pancreatic islets, disturbs the function of GIP in transgenic pigs. Thus we have created a novel, clinically relevant animal model for studying the roles of the GIP/GIPR system. Quantitative morphological studies of the pancreas are being performed to clarify whether GIPR function is essential for pancreatic islet development and maintenance. Reproduction, Fertility and Development 20(1) 82 82 doi:10.1071/RDv20n1Ab4 Diabetes Publish Ahead of Print, published online February 25, 2010 Transgenic pigs with impaired GIP function Glucose intolerance and reduced proliferation of pancreatic b-cells in transgenic pigs with impaired GIP function Simone Renner1, Christiane Fehlings1,*, Nadja Herbach2,*, Andreas Hofmann3, Dagmar C. von Waldthausen1, Barbara Kesler1, Karin Ulrichs5, Irina Chodnevskaja5, Vasiliy Moskalenko5, Werner Amselgruber6, Burkhard Goke7, Alexander Pfeifer3,4, Rudiger Wanke2, Eckhard Wolf1 1Chair for Molecular Animal Breeding and Biotechnology, and Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany 2Institute of Veterinary Pathology, Faculty of Veterinary Medicine, LMU Munich, Veterinarstrasse 13, D-80539 Munich, Germany 3Institute of Pharmacology and Toxicology, and 4Pharma Center Bonn, University of Bonn, Reuterstrasse 2b, D-53113 Bonn, Germany 5Experimental Transplantation Immunology, Surgical Clinic I, University Hospital of Wurzburg, Oberdurrbacher Strasse 6, D-97080 Wurzburg, Germany 6Institute of Anatomy and Physiology, University of Stuttgart-Hohenheim Fruhwirthstrasse 35, D-70593 Stuttgart 7Medical Clinic II, Klinikum Grosshadern, LMU Munich, Marchioninistrasse 15, D-81377 Munich, Germany C.F. and N.H. Contributed equally. Correspondence: Eckhard Wolf, e-mail: ewolf@lmb.uni-muenchen.de Submitted 8 April 2009 and accepted 10 February 2010. Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org Swine Biomedical Models Any manipulation that can be done in mice, can also be accomplished in pigs. Gene knockouts Gene replacement Gene modification Gene addition Modified expression Swine Biomedical Models Any manipulation that can be done in mice, can also be accomplished in pigs. Gene knockouts Gene replacement Gene modification Gene addition Modified expression When the pig is the appropriate model, technology is not an obstacle.