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The pig is an important model organism for biomedical research, mainly due to its extensive genetic, physiological and anatomical similarities with humans. Until date, direct conversion of somatic cells into hepatocyte-like cells (iHeps) has only been achieved in rodents and human cells. Here, we employed lentiviral vectors to screen a panel of 12 hepatic transcription factors (TF) for their potential to convert porcine fibroblasts into hepatocyte-like cells. We demonstrate for the first time, hepatic conversion of porcine somatic cells by over-expression of CEBPα, FOXA1 and HNF4α2 (3TF-piHeps). Reprogrammed 3TF-piHeps display a hepatocyte-like morphology and show functional characteristics of hepatic cells, including albumin secretion, Dil-AcLDL uptake, storage of lipids and glycogen and activity of cytochrome P450 enzymes CYP1A2 and CYP2C33 (CYP2C9 in humans). Moreover, we show that markers of mature hepatocytes are highly expressed in 3TF-piHeps, while fibroblastic markers are reduced. We envision piHeps as useful cell sources for future studies on drug metabolism and toxicity as well as in vitro models for investigation of pig-to-human infectious diseases.

In modern livestock farming horned cattle pose an increased risk of injury for each other as well as for the farmers. Dehorning without anesthesia is associated with stress and pain for the calves and raises concerns regarding animal welfare. Naturally occurring structural variants causing polledness are known for most beef cattle but are rare within the dairy cattle population. The most common structural variant in beef cattle consists of a 202 base pair insertion-deletion (Polled Celtic variant). For the generation of polled offspring from a horned Holstein-Friesian bull, we isolated the Polled Celtic variant from the genome of an Angus cow and integrated it into the genome of fibroblasts taken from the horned bull using the CRISPR/Cas12a system (formerly Cpf1). Modified fibroblasts served as donor cells for somatic cell nuclear transfer and reconstructed embryos were transferred into synchronized recipients. One resulting pregnancy was terminated on day 90 of gestation for the examination of the fetus. Macroscopic and histological analyses proved a polled phenotype. The remaining pregnancy was carried to term and delivered one calf with a polled phenotype which died shortly after birth. In conclusion, we successfully demonstrated the practical application of CRISPR/Cas12a in farm animal breeding and husbandry.

Induced pluripotent stem cells (iPSCs) are a seminal breakthrough in stem cell research and are promising tools for advanced regenerative therapies in humans and reproductive biotechnology in farm animals. iPSCs are particularly valuable in species in which authentic embryonic stem cell (ESC) lines are yet not available. Here, we describe a nonviral method for the derivation of bovine iPSCs employing Sleeping Beauty (SB) and piggyBac (PB) transposon systems encoding different combinations of reprogramming factors, each separated by self-cleaving peptide sequences and driven by the chimeric CAGGS promoter. One bovine iPSC line (biPS-1) generated by a PB vector containing six reprogramming genes was analyzed in detail, including morphology, alkaline phosphatase expression, and typical hallmarks of pluripotency, such as expression of pluripotency markers and formation of mature teratomas in immunodeficient mice. Moreover, the biPS-1 line allowed a second round of SB transposon-mediated gene transfer. These results are promising for derivation of germ line-competent bovine iPSCs and will facilitate genetic modification of the bovine genome.

Porcine induced pluripotent stem cells (iPSCs) are an important animal model for development of regenerative therapies in human medicine. To date, the majority of the porcine cell lines with iPSC characteristics have been generated with the use of viral vectors harboring human or mouse reprogramming factors. Here, we report on the use of Sleeping Beauty transposon vectors based on the porcine transcription factor sequences to reprogram porcine fetal fibroblasts into iPSC-like cells. By using different promoters to drive transgenic expression, we show that the efficiency of reprogramming varies with the promoter type. The cells transfected with two different vector systems under the control of doxycycline-induced tet operator (TetO) promoters failed to upregulate essential endogenous pluripotency genes and to maintain stable iPSC morphology, whereas with the Ef1a and CAG promoters the same vectors proved efficient in generating iPSC-like cells with high levels of endogenous pluripotency gene expression that could be maintained long term in vitro. Our results suggest that the choice of expression vector promoters could significantly influence the efficiency of iPSC production from porcine somatic cells.

The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed at 0.5, 1, 2, 3, 4, 8, 12, and 16 h postactivation (hpa). Parthenogenetic (PA) embryos were used as control. The SCNT and PA embryos were processed for lacmoid staining, autoradiography, transmission electron microscopy (TEM), and immunofluorescence localization of: upstream binding factor (UBF) and fibrillarin at 4 and 12 hpa. Likewise, starved and nonstarved fibroblasts were processed for autoradiography and TEM. The fibroblasts displayed strong transcriptional activity and active fibrillogranular nucleoli. None of the reconstructed embryos, however, displayed transcriptional activity. In conclusion, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development.

Adult stem cells have been previously isolated from a variety of somatic tissues, including bone marrow and the central nervous system; however, contribution of these cells to the germ line has not been shown. Here we demonstrate that fetal somatic explants contain a subpopulation of somatic stem cells (FSSCs), which can be induced to display features of lineage-uncommitted stem cells. After injection into blastocysts, these cells give rise to a variety of cell types in the resultant chimeric fetuses, including those of the mesodermal lineage; they even migrate into the genital ridge. In vitro, FSSCs exhibit characteristics of embryonic stem cells, including extended self-renewal; expression of stem cell marker genes, such as Pou5f1 (Oct4), Stat3, and Akp2 (Tnap) and growth as multicellular aggregates. We report that fetal tissue contains somatic stem cells with greater potency than previously thought, which might form a new source of stem cells useful in somatic nuclear transfer and cell therapy.

Pluripotent embryonic stem cells (ESCs) from the inner cell mass of early murine and human embryos exhibit extensive self-renewal in culture and maintain their ability to differentiate into all cell lineages. These features make ESCs a suitable candidate for cell-replacement therapy. However, the use of early embryos has provoked considerable public debate based on ethical considerations. From this standpoint, stem cells derived from adult tissues are a more easily accepted alternative. Recent results suggest that adult stem cells have a broader range of potency than imagined initially. Although some claims have been called into question by the discovery that fusion between the stem cells and differentiated cells can occur spontaneously, in other cases somatic stem cells have been induced to commit to various lineages by the extra- or intracellular environment. Recent data from our laboratory suggest that changes in culture conditions can expand a subpopulation of cells with a pluripotent phenotype from primary fibroblast cultures. The present paper critically reviews recent data on the potency of somatic stem cells, methods to modify the potency of somatic cells and implications for cell-based therapies.

Enzymes of the Polo-like kinase (Plk) family are active in the pathways controlling mitosis in several species. We have cloned cDNA fragments of the porcine homologues of Plk1, Plk2, and Plk3 employing fetal fibroblasts as source. All three partial cDNAs showed high sequence homology with their mouse and human counterparts and contained the Polo box, a domain characteristic for all Polo kinases. The expression levels of Plk1 mRNA at various points of the cell cycle in synchronized porcine fetal fibroblasts were analyzed by both RT-PCR and the ribonuclease protection assay. Plk1 mRNA was barely detectable in G0 and G1, increased during S phase and peaked after the G2/M transition. A monoclonal antibody was generated against an in vitro expressed porcine Plk1-protein fragment and used to detect changes in Plk1 expression at the protein level. Plk1 protein was first detected by immunoblotting at the beginning of S phase and was highest after the G2/M transition. In summary, the Plk1 expression pattern in the pig is similar to that reported for other species. The absence of Plk1 mRNA and protein appears to be a good marker for G0/G1 and thus for the selection of donor cells for nuclear transfer based somatic cloning., (Copyright 2003 Wiley-Liss, Inc.)

The success of somatic nuclear transfer depends upon the cell cycle stage of the donor nucleus and the recipient cytoplast. Recently, we established efficient cell cycle synchronization protocols for porcine fetal fibroblasts and found that serum withdrawal leads to cell death. Here, we examined whether the specific cell death induced by serum deprivation follows the conventional apoptotic pathway in porcine fibroblasts. Terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling analysis revealed that serum deprivation induced DNA fragmentation in a concentration and time dependent manner. Semiquantitative RT-PCR and Western blotting revealed activation of cell death-related genes Bak and Bax of the Bcl-2 family. However, electrophoretic analysis of genomic DNA from serum deprived cells did not provide evidence for the internucleosomal DNA cleavage which is characteristic of conventional apoptosis. Thus, serum deprivation triggers initial steps in the apoptotic pathway, but does not lead to the typical oligonucleosome-sized DNA ladder. These findings contribute to a better understanding of apoptotic pathways and aid to define essential parameters of the donor nucleus for successful somatic cloning.

Abstract: Background: The programmed cell death‐1 (PD‐1, CD279)/PD‐Ligand1 (PD‐L1, CD274) receptor system is crucial for controlling the balance between immune activation and induction of tolerance via generation of inhibitory signals. Expression of PD‐L1 is associated with reduced immunogenicity and renders cells and tissues to an immune‐privileged/tolerogenic state. Methods: To apply this concept for clinical xenotransplantation, we generated human (h)PD‐L1 transgenic pigs and characterized expression and biological function of the transgene at the cellular level. Results: The hPD‐L1 was detected in kidney, heart, and pancreas. In addition, peripheral blood mononuclear cells (PBMC), cultured fibroblasts, and endothelial cells were hPD‐L1 positive (hPD‐L1+). The hPD‐L1 levels were increased by the treatment of transgenic cells with human cytokines (eg, TNF‐α), suggesting a regulatable mode of transgene expression. Compared to cells from wild‐type pigs, hPD‐L1+ PBMC had a significantly reduced capacity to stimulate proliferation of human CD4+ T cells. Moreover, fibroblasts from hPD‐L1 transgenic pigs were partially protected from cell‐mediated lysis by human cytotoxic effector cells. Conclusions: These data indicate a low immunogenic, immune‐protected status of cells from hPD‐L1 transgenic pigs. The integration of the hPD‐L1 concept into existing multi‐transgenic pigs is promising to achieve long‐term survival of porcine xenografts in non‐human primate recipients. [ABSTRACT FROM AUTHOR]

Semaan M, Kaulitz D, Petersen B, Niemann H, Denner J. Long-term effects of PERV-specific RNA interference in transgenic pigs. Xenotransplantation 2012; 19: 112-121. © 2012 John Wiley & Sons A/S. Abstract: Background: Porcine endogenous retroviruses (PERVs) represent a risk of xenotransplantation using porcine cells, tissues, or organs, as they are integrated in the porcine genome and have been shown to be able to infect human cells in vitro. To increase viral safety by RNA interference, transgenic pigs expressing a PERV-specific small hairpin (sh)RNA targeted to a highly conserved sequence in the pol gene (pol2) were generated in which expression of PERVs was reduced (Xenotransplantation, 15, 2008, 38). However, it remains to be shown how long expression of the shRNA and the RNA interference is effective in reducing PERV expression. Methods: To analyze the long-term duration of RNA interference, expression of the PERV-specific pol2 shRNA and inhibition of PERV expression was studied repeatedly in fibroblasts and peripheral blood mononuclear cells (PBMCs) of transgenic pigs over a period of 3 yr, when animals were sacrificed and expression was studied in different organs. Expression of the PERV-specific shRNA was measured using a newly developed real-time PCR, and expression of PERV was measured using a PERV-specific real-time PCR. Results: Over a period of 3 yr, PERV-specific shRNA and green fluorescent protein (GFP) as reporter of the vector system were consistently expressed in transgenic animals. PERV expression was significantly reduced during the entire period. Levels of PERV and shRNA expression were different in the various organs. PERV expression was highest in the spleen and the lungs and lowest in liver and heart. However, in all organs of the transgenic pigs, PERV expression was inhibited compared with the vector control animals. Conclusions: Transgenic pigs expressing PERV-specific shRNA maintained their specific RNA interference long term, suggesting that PERV expression in the xenotransplants will be suppressed over extended periods of time. [ABSTRACT FROM AUTHOR]

Petersen B, Ramackers W, Tiede A, Lucas-Hahn A, Herrmann D, Barg-Kues B, Schuettler W, Friedrich L, Schwinzer R, Winkler M, Niemann H. Pigs transgenic for human thrombomodulin have elevated production of activated protein C. Xenotransplantation 2009; 16: 486–495. © 2009 John Wiley & Sons A/S. 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/105 cells/2h) was significantly higher in fibroblasts of hTM-transgenic clones compared to wild-type porcine fibroblasts (1.7 U/105 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. [ABSTRACT FROM AUTHOR]

Background: Xenotransplantation using porcine cells, tissues or organs may be associated with the transmission of porcine endogenous retroviruses (PERVs). More than 50 viral copies have been identified in the pig genome and three different subtypes of PERV were released from pig cells, two of them were able to infect human cells in vitro. RNA interference is a promising option to inhibit PERV transmission. Methods: We recently selected an efficient si (small interfering) RNA corresponding to a highly conserved region in the PERV DNA, which is able to inhibit expression of all PERV subtypes in PERV-infected human cells as well as in primary pig cells. Pig fibroblasts were transfected using a lentiviral vector expressing a corresponding sh (short hairpin) RNA and transgenic pigs were produced by somatic nuclear transfer cloning. Integration of the vector was proven by PCR, expression of shRNA and PERV was studied by in-solution hybridization analysis and real-time RT PCR, respectively. Results: All seven born piglets had integrated the transgene. Expression of the shRNA was found in all tissues investigated and PERV expression was significantly inhibited when compared with wild-type control animals. Conclusion: This strategy may lead to animals compatible with PERV safe xenotransplantation. [ABSTRACT FROM AUTHOR]

During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7-gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene (GMNN). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9-GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9-GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches. [ABSTRACT FROM AUTHOR]

Background Pig-to-human xenotransplantation is promising to ameliorate the worldwide shortage of suitable human donor organs. The transplantation of xenografts evokes severe immunological rejection responses. Different approaches led to considerable progress in this respect: transgenic expression of human complement regulatory proteins such as human decay-accelerating factor (hCD55), membrane co-factor protein (hCD46, MCP) or hCD59 and/or knockout of the porcine α1,3-galactosyltransferase gene (GGTA1-KO) prevent the hyperacute rejection response (HAR) [1, 2]. Moreover, human anticoagulant and/or anti-apoptotic and anti-inflammatory transgenes, such as human A20 (hA20) and human heme oxygenase-1 (hHO-1), could protect porcine xenografts from being rejected by the acute vascular rejection response [3, 4]. Recently, we produced pigs transgenic for hA20 which provides cytoprotective properties in porcine aortic endothelial cells in vitro [5]. However, hA20 was expressed only at low levels in heart, skeletal muscle and porcine aortic endothelial cells. Here, we generated a new tri-cistronic hA20/hCD55/Puro vector based on the Sleeping Beauty system (kindly provided by Dr. Zoltán Ivics) and transfected it into porcine GGTA1-KO cells that served as donor cells for somatic cell nuclear transfer (SCNT). Methods We co-transfected GGTA1-KO [6] porcine fibroblasts with a tri-cistronic cDNA expression vector coding for hA20/hCD55/Puro and the SB transposase 100X plasmid. The advantage of this vector is a stable genomic integration without any plasmid-based vector backbone and the combined integration of all transgenes at a single genomic locus which avoids transgene segregation after mating. Following puromycin selection (5 μg/ml), cells were screened by PCR for genomic integration of the vector and used for SCNT. Results In total, 1,028 embryos were transferred to 13 recipients of which five remained pregnant. Two pregnancies were in too early stages for ultrasound diagnosis at the time of writing. One pregnancy was terminated on day 25 of gestation and eight fetuses were obtained. The hA20 mRNA levels of these fetuses were increased 21- to 44-fold compared to another line (hA20/hHO-1/GGTA1-KO) and even higher than in the first generation hA20 transgenic pigs. Preliminary immunofluorescence analysis data showed delta mean fluorescence intensities (MFI) for hCD55 (hCD55 MFI minus control antibody MFI) of 1,016 in average in transgenic fibroblasts in comparison to delta MFI 299 in HUVECs and 114 in PBMCs. The susceptibility of the cells to NK cell- and T cell-mediated cell death compared to wild-type fibroblasts will be determined in a 51chromium release assay. Three gilts are expected to deliver piglets in November of this year. The expression and organ distribution of hA20 and hCD55 will be characterised subsequently. Conclusions Our goal is the production of multi-transgenic pigs with an increased expression of xeno-relevant transgenes. The combination of ubiquitous, high expression of anti-apoptotic and-inflammatory transgenes such as hA20 together with a complement regulatory protein like hCD55 on a GGTA1-KO background has the potential to prolong the survival of porcine grafts after pig-to-primate xenotransplantation. References [1] Phelps CJ, Koike C, Vaught TD, et al. Production of alpha 1,3-Galactosyltransferase-Deficient Pigs. Science. 2002; 299: 411-4. [2] Cowan PJ, Aminan A, Barlow H, et al. Renal xenografts from triple-transgenic pigs are not hyperacutely rejected but cause coagulopathy in non-immunosuppressed baboons. Transplantation. 2000; 69: 2504-2515. [3] Bach FH, Ferran C, Hechenleitner P, et al. Accommodation of vascularized xenografts: expression of 'protective genes' by donor endothelial cells in a host Th2 cytokine environment. Nat Med. 1997; 3: 196-204. [4] Petersen B, Ramackers W, Lucas-Hahn A, et al. Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys. Xenotransplantation 2011; 18: 355-368. [5] Oropeza M, Petersen B, Carnwerth J W, et al. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli. Xenotransplantation 2009; 16: 522-534. [6] Hausschild J, Peterssen B, Santiago Y, et al. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 2011; 108: 12013-12027. [ABSTRACT FROM AUTHOR]

Multi-transgenic pigs are required for prolonging survival of porcine xenografts after transplantation into primate recipients. A homozygous knockout of the alpha1,3-galactosyltransferase gene (GGTA-1, encoding for Gal-epitopes) is critical for controlling the hyperacute rejection response. Nevertheless, the additional expression of complement regulatory proteins such as CD55 (decay accelerating factor, DAF) on a GGTA-1 KO background is thought to further improve survival after xenotransplantation. The next hurdle is the acute vascular rejection (AVR), which is primarily caused by endothelial cell activation. Previous experiments had shown that expression of anti-apoptotic/anti-inflammatory genes, such as human heme oxygenase-1 (hHO-1) or human A20 in pig organs may overcome AVR (1, 2). Adult fibroblasts from previously generated hHO-1 transgenic pigs were transfected with Zinc-finger nuclease plasmids directed towards knockout of the GGTA-1 gene. Transfected cells were selected via a magnetic bead selection approach as previously described (3). GGTA-1-KO/hHO-1 cells were used as donor cells in somatic cell nuclear transfer (SCNT). The first pregnancy was terminated at day 25 of gestation and 11 healthy fetuses were obtained. All fetuses were GGTA-1-KO and expressed hHO-1 at similar levels as the hHO-1 transgenic cell donors. Next, fetal GGTA-1-KO/hHO-1 cells from one fetus (C6F5) were transfected with a Sleeping Beauty (SB) transposon (kindly provided by Dr. Zoltan Ivics) coding for hCD55. Two positive cell clones were pooled and used as donor cells for SCNT. One pregnancy was terminated on day 25 and four healthy fetuses were obtained which showed the desired GGTA-1 KO/HO-1/CD55 genotype. Real-time PCR detected a 0.9 fold expression of CD55 over the housekeeping gene GAPDH. Compared to human umbilical vein cells (HUVEC), GGTA-1 KO/HO-1/CD55 fetal fibroblasts expressed CD55 10-15 fold higher than HUVEC. Two fetuses showed a slightly higher expression of CD55 and are currently used as cell donors for recloning to produce live triple transgenic offspring. To solve the problem of transgene segregation occurring after mating of multitransgenic pigs, we generated two tri-cistronic SB vectors coding for A20 and CD55 separated by the self-cleaving peptide E2 site. To select for transfected cells, one vector carried a Tomato-fluorescence cassette whereas the second conferred puromycin resistance. Red fluorescent/puromycin resistant cells served as donor cells for SCNT. Six healthy fetuses were collected on day 25 of pregnancy; all showed high expression of A20 and CD55 as detected by Real-time PCR and FACS. One of the fetuses was used as cell donor for recloning. Currently, three pregnancies with A20/CD55 fetuses are going on and will be allowed to go to term. These results show that molecular scissors, including ZFNs and TALENS, and expression vectors such as the Sleeping beauty system are valuable tools to enhance and accelerate the generation of multi-transgenic pigs for xenotransplantation. References [1] Oropeza M, Petersen B, Carnwath JW et al. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli. Xenotransplantation. Proc. Nat. Acad. Sci. USA 2009; 16: 522-534. [2] Petersen B, Ramackers W, Lucas-Hahn A et al. Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys. Xenotransplantation 2011; in press. [3] Hauschild J, Petersen B, Santiago Y et al. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc. Nat. Acad. Sci. USA. 2011; 108: 12013-12017. [ABSTRACT FROM AUTHOR]