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Vertebrate reproductive science and technology
RESEARCH ARTICLE

309 PRODUCTION OF PIGS TRANSGENIC FOR HUMAN HEMEOXYGENASE-I BY SOMATIC NUCLEAR TRANSFER

B. Petersen, A. Lucas-Hahn, D. Herrmann, W. A. Kues, W. Ramackers, S. Bergmann, J. W. Carnwath, M. Winkler and H. Niemann

Reproduction, Fertility and Development 20(1) 234 - 234
Published: 12 December 2007

Abstract

The hyperacute rejection after porcine-to-human xenotransplantation can now be reliably overcome either by transgenic expression of complement regulating factors or by knocking out the gene for α1,3-galactosyltransferase in pigs. The next immunological hurdle is the acute vascular rejection (AVR) primarily caused by endothelial cell activation. Human hemeoxygenase-I (hHO-I) is known to have anti-apoptotic and cell-protective properties. Thus, the expression of hHO-I on porcine endothelial cells could have beneficial effects in a xenotransplantation setting to prevent the formation of AVR. Here we describe the first transgenic pigs with functional expression of hHO-I. Fibroblasts were obtained by an ear punch from a decay accelerating factor (DAF)-transgenic female pig and were cultured in vitro (Kues WA et al. 2005 Biol. Reprod. 72, 1020–1028). Cells reaching confluency of 70 to 80% were detached with EDTA/trypsin and subsequently transfected by electroporation at 450V/350 µF with a vector coding for hHO-I driven by the SV40 promoter. Transfected cells were selected for resistance against G418 (800 µg mL–1) for 14 days. Resistant cell clones were screened for integration of the vector by PCR. One positive cell clone that showed strong expression of the transgene, as determined by immunostaining, was selected for somatic nuclear transfer (Hoelker M et al. 2005 Cloning Stem Cells 7, 35–44). In total, 205 reconstructed embryos were transferred to 2 synchronized peripuberal German Landrace gilts, which gave birth to 9 live piglets, all with normal birth weights. PCR and Southern blot analyses revealed that all of the offspring had integrated the vector into their genome. Two transgenic animals were sacrificed for further characterization and ex vivo perfusion experiments. In these animals, Northern blotting showed weak transcription of the transgene in all xenorelevant organs such as heart, kidney, and liver. Immunostaining of the kidneys revealed that expression of the transgene was confined to the endothelial cell layer. These hHO-I-transgenic porcine kidneys were subjected to an ex vivo perfusion assay and survived ex vivo perfusion for 240 min with AB-pooled human blood, whereas perfusion of 2 non-transgenic controls had to be terminated after 60 min due to problems attributed to immunological rejection. Histology revealed that perfused hHO-I-transgenic kidneys exhibited no indication for xenogenic activation of the human coagulation system. These preliminary results show that functional hHO-I can be expressed in transgenic pigs and that transgenic expression of hHO-I might improve long-term survival of porcine xenografts. These results are encouraging and warrant further studies on endothelial cell activation and the biological function of hemeoxygenase-I in the context of xenotransplantation.

This study was funded by the Deutsche Forschungsgemeinschaft Ni 256/22-1, -2, -3.

https://doi.org/10.1071/RDv20n1Ab309

© CSIRO 2007

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