Production and characterization of pigs transgenic for human hemeoxygenase‐I by somatic nuclear transfer

Abstract

Introduction: The hyperacute rejection after porcine‐to‐human xenotransplantation can now be reliably overcome either by transgenic expression of complement regulating factors (done in this study) 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‐1) is known to have 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 describe the generation and characterization of pigs transgenic for hHO‐1.Methods: Fibroblasts were obtained by an ear punch from a CD55 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 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 selected for somatic nuclear transfer.In total, 205 reconstructed embryos were transferred to two synchronized peripuberal German Landrace gilts which gave birth to nine live piglets, all with normal birth weights. The expression of the transgenes (CD55 and hHO‐1) was determined by flow cytometry and Western blot in endothelial cells and peripheral blood lymphocytes. The expression pattern of the transgenes in different organs was shown by immunostaining, RT‐PCR and Northern blot. Organ survival and XAC following the contact of human blood with porcine endothelium, were determined by using an ex‐vivo perfusion circuit based on low‐dose heparin mediated anticoagulation. Porcine kidneys were recovered following in situ cold perfusion with HTK organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled human AB blood.Results: PCR and Southern blot analyses revealed that all of the offspring had integrated the vector in their genome. Six transgenic animals were sacrificed for further characterization. In these animals, the expression of CD55 and hHO‐1 in peripheral blood lymphocytes and cultured endothelial cells was shown by flow cytometry and Western blot. In addition, albeit all animals were cloned from the same cell clone, variations in the expression pattern and levels could be observed in immunostaining, RT‐PCR and Northern Blot. The animals showed only weak expression of hHO‐1 in most of the xenorelevant organs like heart, kidney and liver. Average survival of wildtype pig kidneys during organ perfusion with human blood and addition of heparin was 42 ± 26 min. Application of soluble complement inhibition (C1‐inhibitor) prolonged organ survival to 126 ± 72 min. XAC was observed with significantly elevated concentrations of d‐Dimer and thrombin antithrombin complex (TAT) combined with consumption of fibrinogen and antithrombin. The histological analyses revealed numerous microthrombi. In contrast, the perfusion of the CD55/hHO1 transgenic porcine kidneys was feasible for more than 240 min in all perfusion experiments (with and without C1‐inh). Although elevated levels of d‐Dimer and TAT were measured, no significant consumption of fibrinogen and antithrombin occurred. In addition, no microthrombi were detectable histologically.Conclusion: These results show that hHO‐1 in combination with CD55 can be expressed in transgenic pigs. Somatic cell nuclear transfer is a powerful tool for the generation of transgenic animals for xenotransplantation. 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.