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Generation and characterization of pigs transgenic for human hemeoxygenase‐1 (hHO‐1)

Generation and characterization of pigs transgenic for human hemeoxygenase‐1 (hHO‐1) Introduction: The hyperacute rejection after porcine‐to‐human xenotransplantation can now be reliably overcome either by transgenic expression of human complement regulating factors or by knocking out the gene for α(1,3)‐galactosyltransferase in pigs. The next immunological hurdle is the acute vascular rejection (AVR) primarily caused by endothelial cell activation. Human hemeoxygenase‐I (hHO‐1) has anti‐apoptotic and cell protective properties. Thus, the expression of hHO‐1 on porcine endothelial cells could have beneficial effects in a xenotransplantation setting. Here, we report the generation and characterization of pigs transgenic for hHO‐1. Methods: Fibroblasts were obtained by an ear punch from a female pig that showed a mosaic expression of hDAF and were cultured in vitro as described previously indoleamine 2,3‐dioxygenase (1). Cells reaching confluency of 70–80%, were detached with EDTA/trypsin and subsequently transfected by electroporation at 450 V/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) for 14 days. Resistant cell clones were screened for integration of the vector by PCR. One positive cell clone was used in somatic nuclear transfer. In total, 205 reconstructed embryos were transferred to two synchronized peripubertal German Landrace gilts which gave birth to nine live piglets with normal birth weights. The integration and expression of hHO‐1 and hDAF were determined by PCR, Southern blot and RT‐PCR. Cell cultures were established from a hHO‐1 transgenic piglet with no DAF‐integrant. Cells were used as donor cells in a recloning approach to produce a homogenous group of hHO‐1 transgenic animals and eleven genetically hHO‐1 transgenic offspring were obtained. Expression of hHO‐1 was determined in different organs by RT‐PCR, Northern and Western blot; and in endothelial cells and peripheral blood lymphocytes by flow cytometry and Western blot. Resistance of hHO‐1 transgenic PAECs against TNF‐α mediated (1 ng, 10 ng, 30 ng, 50 ng TNF‐α) apoptosis was detected with a caspase GLO assay (Promega, Germany) in a luminometer. Endothelial cell activation was measured by realtime PCR using primers for the adhesion molecules ICAM‐1, VCAM‐1 and E‐selectin. Results: PCR and Southern blot analyses revealed that all of offspring had integrated the vector in their genome. Six transgenic animals were sacrificed for in depth characterization. However, albeit all animals were cloned from the same cell clone, variation in the expression pattern and levels was observed in RT‐PCR and Northern Blot. The recloned animals showed identical expression levels and patterns of hHO‐1. All animals showed weak expression of hHO‐1 in most of the xenorelevant organs like heart, kidney and liver. HO‐1 transgenic PAECs were significantly resistant to TNF‐α mediated apoptosis compared to wild type PAECs. To show that this protection was due to hHO‐1 over‐expression, transgenic cells were incubated with the specific hHO‐1 inhibitor ZnPP IX (20 μM). Transgenic PAECs were not protected against TNF‐α mediated apoptosis in the presence of ZnPPIX. Realtime PCR revealed reduced expression of adhesion molecules (ICAM‐1,VCAM‐1,E‐selectin) after TNF‐α treatment in hHO‐1 transgenic PAECs compared to wild type controls. This effect could be inhibited by incubation of transgenic PAECs with ZnPP IX. HHO‐1 transgenic kidneys were tested in an ex vivo perfusion circuit (2). Preliminary results show that hHO‐1 transgenic kidneys could be perfused for the maximum perfusion time of 240 min and microthrombi were not detected histologically. Conclusion: Somatic cell nuclear transfer is a powerful tool for the production of transgenic animals for xenotransplantation. The HHO‐1 can be functionally expressed in transgenic pigs and is a promising candidate gene to prevent acute vascular rejection or to minimize damage after ischemia/reperfusion. 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,‐4. References 1. Kues WA , Petersen B, Mysegades W et al. Isolation of murine and porcine fetal stem cells from somatic tissue. Biol Reprod 2005; 72: 1020–1028. 2. Ramackers W, Friedrich L,Tiede A. Effects of pharmacological intervention on coagulopathy and organ function in xenoperfused kidneys. Xenotransplantation 2008; 15: 46–55. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Xenotransplantation Wiley

Generation and characterization of pigs transgenic for human hemeoxygenase‐1 (hHO‐1)

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References (2)

Publisher
Wiley
Copyright
© 2010 John Wiley & Sons A/S
ISSN
0908-665X
eISSN
1399-3089
DOI
10.1111/j.1399-3089.2010.00573_7.x
Publisher site
See Article on Publisher Site

Abstract

Introduction: The hyperacute rejection after porcine‐to‐human xenotransplantation can now be reliably overcome either by transgenic expression of human complement regulating factors or by knocking out the gene for α(1,3)‐galactosyltransferase in pigs. The next immunological hurdle is the acute vascular rejection (AVR) primarily caused by endothelial cell activation. Human hemeoxygenase‐I (hHO‐1) has anti‐apoptotic and cell protective properties. Thus, the expression of hHO‐1 on porcine endothelial cells could have beneficial effects in a xenotransplantation setting. Here, we report the generation and characterization of pigs transgenic for hHO‐1. Methods: Fibroblasts were obtained by an ear punch from a female pig that showed a mosaic expression of hDAF and were cultured in vitro as described previously indoleamine 2,3‐dioxygenase (1). Cells reaching confluency of 70–80%, were detached with EDTA/trypsin and subsequently transfected by electroporation at 450 V/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) for 14 days. Resistant cell clones were screened for integration of the vector by PCR. One positive cell clone was used in somatic nuclear transfer. In total, 205 reconstructed embryos were transferred to two synchronized peripubertal German Landrace gilts which gave birth to nine live piglets with normal birth weights. The integration and expression of hHO‐1 and hDAF were determined by PCR, Southern blot and RT‐PCR. Cell cultures were established from a hHO‐1 transgenic piglet with no DAF‐integrant. Cells were used as donor cells in a recloning approach to produce a homogenous group of hHO‐1 transgenic animals and eleven genetically hHO‐1 transgenic offspring were obtained. Expression of hHO‐1 was determined in different organs by RT‐PCR, Northern and Western blot; and in endothelial cells and peripheral blood lymphocytes by flow cytometry and Western blot. Resistance of hHO‐1 transgenic PAECs against TNF‐α mediated (1 ng, 10 ng, 30 ng, 50 ng TNF‐α) apoptosis was detected with a caspase GLO assay (Promega, Germany) in a luminometer. Endothelial cell activation was measured by realtime PCR using primers for the adhesion molecules ICAM‐1, VCAM‐1 and E‐selectin. Results: PCR and Southern blot analyses revealed that all of offspring had integrated the vector in their genome. Six transgenic animals were sacrificed for in depth characterization. However, albeit all animals were cloned from the same cell clone, variation in the expression pattern and levels was observed in RT‐PCR and Northern Blot. The recloned animals showed identical expression levels and patterns of hHO‐1. All animals showed weak expression of hHO‐1 in most of the xenorelevant organs like heart, kidney and liver. HO‐1 transgenic PAECs were significantly resistant to TNF‐α mediated apoptosis compared to wild type PAECs. To show that this protection was due to hHO‐1 over‐expression, transgenic cells were incubated with the specific hHO‐1 inhibitor ZnPP IX (20 μM). Transgenic PAECs were not protected against TNF‐α mediated apoptosis in the presence of ZnPPIX. Realtime PCR revealed reduced expression of adhesion molecules (ICAM‐1,VCAM‐1,E‐selectin) after TNF‐α treatment in hHO‐1 transgenic PAECs compared to wild type controls. This effect could be inhibited by incubation of transgenic PAECs with ZnPP IX. HHO‐1 transgenic kidneys were tested in an ex vivo perfusion circuit (2). Preliminary results show that hHO‐1 transgenic kidneys could be perfused for the maximum perfusion time of 240 min and microthrombi were not detected histologically. Conclusion: Somatic cell nuclear transfer is a powerful tool for the production of transgenic animals for xenotransplantation. The HHO‐1 can be functionally expressed in transgenic pigs and is a promising candidate gene to prevent acute vascular rejection or to minimize damage after ischemia/reperfusion. 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,‐4. References 1. Kues WA , Petersen B, Mysegades W et al. Isolation of murine and porcine fetal stem cells from somatic tissue. Biol Reprod 2005; 72: 1020–1028. 2. Ramackers W, Friedrich L,Tiede A. Effects of pharmacological intervention on coagulopathy and organ function in xenoperfused kidneys. Xenotransplantation 2008; 15: 46–55.

Journal

XenotransplantationWiley

Published: Mar 1, 2010

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