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The domestic pig shares many genetic, anatomical and physiological similarities to humans and is thus considered to be a suitable organ donor for xenotransplantation. However, prior to clinical application of porcine xenografts, three major hurdles have to be overcome: (1) various immunological rejection responses, (2) physiological incompatibilities between the porcine organ and the human recipient and (3) the risk of transmitting zoonotic pathogens from pig to humans. With the introduction of genetically engineered pigs expressing high levels of human complement regulatory proteins or lacking expression of α-Gal epitopes, the HAR can be consistently overcome. However, none of the transgenic porcine organs available to date was fully protected against the binding of anti-non-Gal xenoreactive natural antibodies. The present view is that long-term survival of xenografts after transplantation into primates requires additional modifications of the porcine genome and a specifically tailored immunosuppression regimen compliant with current clinical standards. This requires the production and characterization of multi-transgenic pigs to control HAR, AVR and DXR. The recent emergence of new sophisticated molecular tools such as Zinc-Finger nucleases, Transcription-activator like endonucleases, and the CRISPR/Cas9 system has significantly increased efficiency and precision of the production of genetically modified pigs for xenotransplantation. Several candidate genes, incl. hTM, hHO-1, hA20, CTLA4Ig, have been explored in their ability to improve long-term survival of porcine xenografts after transplantation into non-human primates. This review provides an update on the current status in the production of multi-transgenic pigs for xenotransplantation which could bring porcine xenografts closer to clinical application.

Immunisation of goats, mice, rats, rabbits, guinea pigs, and hamsters with the recombinant ectodomain of the porcine endogenous retrovirus (PERV) transmembrane envelope (TM) protein (p15E) induced binding and neutralising immune antibodies in all animals. In contrast, no antibodies were induced when pigs were immunised with p15E, indicating that pigs are tolerant to their endogenous retroviruses, at least to the TM protein. To answer the question of whether pigs are tolerant to other structural proteins of PERV, we immunised German landrace pigs with p15E, this time in conjunction with the surface envelope proteins gp70 and the core capsid Gag protein p27CA. To ensure that the pigs were immunocompetent and that immunisation was successful, all animals also received an injection of an unrelated protein, keyhole limpet hemocyanin (KLH). Whereas all animals produced antibodies against KLH, no animals produced antibodies against the viral envelope proteins, thus confirming previous results for p15E and extending them to the other envelope protein, gp70. However, the pigs did produce antibodies against p27CA, indicating that there is no tolerance to the core capsid protein of PERV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In vitro production of porcine embryos by means of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) is limited by great inefficienciy. The present study investigated chromatin and nucleolar dynamics in porcine embryos developed in vivo (IV) and compared this physiological standard to that of embryos produced by IVF, parthenogenetic activation (PA), or SCNT. In contrast to IV embryos, chromatin spatial and temporal dynamics in PA, IVF, and SCNT embryos were altered; starting with aberrant chromatin-nuclear envelope interactions at the two-cell stage, delayed chromatin decondensation and nucleolar development at the four-cell stage, and ultimately culminating in failure of proper first lineage segregation at the blastocyst stage, demonstrated by poorly defined inner cell mass. Interestingly, in vitro produced (IVP) embryos also lacked a heterochromatin halo around nucleolar precursors, indicating imperfections in global chromatin remodeling after fertilization/activation. Porcine IV-produced zygotes and embryos display a well-synchronized pattern of chromatin dynamics compatible with genome activation and regular nucleolar formation at the four-cell stage. Production of porcine embryos under in vitro conditions by IVF, PA, or SCNT is associated with altered chromatin remodeling, delayed nucleolar formation, and poorly defined lineage segregation at the blastocyst stage, which in turn may impair their developmental capacity.

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., (© 2012 John Wiley & Sons A/S.)

DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome. Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts despite the occurrence of de novo methylation in early blastocysts. Comparable levels of DNA methylation were found in IV embryos and in 50% and 14% of SCNT early and late blastocysts, respectively. In conclusion, DNA methylation patterns were adversely affected by in vitro embryo production.

Genetic engineering can expand the utility of pigs for modeling human diseases, and for developing advanced therapeutic approaches. However, the inefficient production of transgenic pigs represents a technological bottleneck. Here, we assessed the hyperactive Sleeping Beauty (SB100X) transposon system for enzyme-catalyzed transgene integration into the embryonic porcine genome. The components of the transposon vector system were microinjected as circular plasmids into the cytoplasm of porcine zygotes, resulting in high frequencies of transgenic fetuses and piglets. The transgenic animals showed normal development and persistent reporter gene expression for >12 months. Molecular hallmarks of transposition were confirmed by analysis of 25 genomic insertion sites. We demonstrate germ-line transmission, segregation of individual transposons, and continued, copy number-dependent transgene expression in F1-offspring. In addition, we demonstrate target-selected gene insertion into transposon-tagged genomic loci by Cre-loxP-based cassette exchange in somatic cells followed by nuclear transfer. Transposase-catalyzed transgenesis in a large mammalian species expands the arsenal of transgenic technologies for use in domestic animals and will facilitate the development of large animal models for human diseases.

Background: 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/10(5) cells/2h) was significantly higher in fibroblasts of hTM-transgenic clones compared to wild-type porcine fibroblasts (1.7 U/10(5) 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.

The shortage of donated human organs for transplantation continues to be a life threatening problem for patients suffering from complete organ failure. Although this gap is increasing due to the demographic changes in aging Western populations, it is generally accepted that international trading in human organ is not an ethical solution. Alternatives to the use of human organs for transplantation must be developed and these alternatives include stem cell therapy, artificial organs and organs from other species, i.e. xenografts. For practical reasons but most importantly because of its physiological similarity with humans, the pig is generally accepted as the species of choice for xenotransplantation. Nevertheless, before porcine organs can be used in human xenotransplantation, it is necessary to make a series of precise genetic modifications to the porcine genome, including the addition of genes for factors which suppress the rejection of transplanted porcine tissues and the inactivation or removal of undesirable genes which can only be accomplished at this time by targeted recombination and somatic nuclear transfer. This review will give an insight into the advances in transgenic manipulation and cloning in pigs--in the context of porcine-to-human xenotransplantation.

The effects of solid surface vitrification (SSV) on viability and parthenogenetic development of in vitro matured (IVM) porcine oocytes was investigated in the present study. Cumulus-free IVM porcine oocytes were subjected either to SSV or SSV combined with a cytochalasin B (CB) pre-treatment (SSV+CB) or all steps of SSV but without cooling (toxicity control=TC; toxicity control with CB pre-treatment=TC+CB). Oocyte viability was evaluated by plasma membrane integrity and esterase activity measured by a combined staining with fluorescein diacetate, propidium iodide and Hoechst 33342. Surviving oocytes were parthenogenetically activated then cultured in vitro (IVC) for 6 days. The proportion of live oocytes after vitrification was significantly lower than that of the TC, TC+CB and the control groups, regardless of the CB pre-treatment. Treatment of oocytes with cryoprotectants did not decrease the rates of surviving oocytes. After activation of oocytes, the proportion of cleaved embryos was significantly higher in the SSV+CB (P<0.05) than that of the SSV group. Nevertheless, significantly more oocytes cleaved (P<0.05) in the TC, TC+CB and the control groups. On Day 6, the rate of blastocysts in the SSV and SSV+CB groups did not differ significantly. The number of oocytes developing to blastocyst and the mean number of blastomeres per embryo were significantly higher (P<0.05) in the TC, TC+CB and the control compared with that of the SSV and SSV+CB groups. To our knowledge, this is the first report on parthenogenetic development to blastocysts of porcine oocytes vitrified at the metaphase-II stage. Results indicate that the high concentrations of cryoprotectants were not harmful for in vitro development, and that CB pre-treatment may increase survival and development of SSV vitrified porcine oocytes.

The applicability of tightly regulated transgenesis in domesticated animals is severely hampered by the present lack of knowledge of regulatory mechanisms and the long generation intervals. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single-step transduction to introduce an autoregulative tetracycline-responsive bicistronic expression cassette (NTA) into transgenic pigs. Transgenic pigs carrying one NTA cassette showed a mosaic transgene expression restricted to single muscle fibers. In contrast, crossbred animals carrying two NTA cassettes with different transgenes, revealed a broad tissue-independent and tightly regulated expression of one cassette, but not of the other one. The expression pattern correlated inversely with the methylation status of the NTA transcription start sites indicating epigenetic silencing of one NTA cassette. This first approach on tetracycline regulated transgene expression in farm animals will be valuable for developing precisely controlled expression systems for transgenes in large animals relevant for biomedical and agricultural biotechnology.

Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.

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]

Background: Interventional endoscopies entail a risk of infection secondary to perforation of the luminal wall. Thereby, bacteria may be introduced into the sterile environment of the peritoneal cavity (PC). Limited data are available regarding the efficacy of prophylactic anti-infective treatments. The aim of the study was to examine the efficacy/safety of anti-infective means in the prevention of infection by interventional endoscopies in a randomized controlled animal trial.Methods: Forty pigs were randomized to: 1: control; 2: oral lavage; 3: gastric lavage; 4: oral/gastric lavage; 5: i.m. antibiotics. Lavage was performed with Octenisept prior to the operation. After gastric wall perforation, peritoneoscopy was performed. Before the procedure, after closure and prior to autopsy, intraabdominal lavage for bacterial culture was taken using mini-laparoscopy. At autopsy, macroscopic appearance of the PC was scored. Lavage fluids were grown to identify/quantify bacterial load. Concentration of intraperitoneal bacteria at autopsy was defined as main outcome parameter.Results: No major complications occurred in any of the procedures. Bacterial load of the PC at autopsy was significantly reduced with antibiotics compared to all other groups, whereas it did not differ between the lavage groups and control. Macroscopic scoring of the PC showed significant lower rate of intraabdominal abscesses in the antibiotic group compared to the lavage groups and control (p < 0.01).Conclusion: Only antibiotic prophylaxis is effective for the prevention of infection after iatrogenic perforation of the gastrointestinal wall. There was no difference between any form of lavage and the control group. Further studies in humans are required to prove these animal data. [ABSTRACT FROM AUTHOR]

Oropeza M, Petersen B, Carnwath JW, Lucas-Hahn A, Lemme E, Hassel P, Herrmann D, Barg-Kues B, Holler S, Queisser A-L, Schwinzer R, Hinkel R, Kupatt C, Niemann H. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli. Xenotransplantation 2009; 16: 522–534. © 2009 John Wiley & Sons A/S. Porcine organs with transgenic expression of anti-apoptotic and anti-inflammatory genes like the human A20 gene (hA20), a tumor necrosis factor-alpha (TNF-α)-inducible gene, may control the acute vascular rejection (AVR) of porcine xenografts. The human A20 molecule possesses protective features against inflammatory and apoptotic stimuli in various cell types including endothelial cells, rendering it a promising candidate for transgenic pig production in the context of xenotransplantation. Here, we produced pigs transgenic for hA20 and investigated whether hA20-transgenic porcine aortic endothelial cells (PAECs) were resistant against the induction of apoptosis in vitro and to what extent hA20-transgenic porcine hearts were protected against ischemia/reperfusion (I/R) injury. Methods: Porcine fetal fibroblasts (PFFs) were transfected with the vector pCAGGSEhA20-IRESNEO containing a chicken β-actin/rabbit β-globin (CAGGS)-promoter element, known to provide ubiquitous gene expression in both mice and pigs. Transfected PFFs were then used in somatic cell nuclear transfer (SCNT). Three hA20-transgenic pigs were killed for PAEC isolation and organ mRNA and protein expression analysis by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern and Western Blotting. PAECs were tested for susceptibility to apoptosis after TNF-α challenging and triggering of the CD95(Fas)/CD95Ligand pathway. Five transgenic and three wild type animals were subjected to an I/R experiment followed by measurement of infarct size, myeloperoxidase (MPO) activity and subendocardial segmental shortening (SES) to assess protective effects of hA20 in the porcine myocardium. Results: The hA20-transgenic pigs developed normally and expression of hA20 was found in skeletal muscle, heart and PAECs. Cultured human A20-transgenic PAECs showed significantly reduced apoptosis when compared to their wild type counterparts and were less susceptible to the induction of cell death by CD95(Fas)L. Only partial protection of hA20-transgenic pig hearts was observed after I/R. While infarct size did not differ between the two groups after ischemic assault, hA20-transgenic porcine hearts showed significantly lower MPO activity and better hemodynamic performance (determined as SES) than their wild type counterparts. Conclusions: The hA20 gene was for the first time functionally expressed in transgenic pigs. Although the CAGGS is a ubiquitous promoter element, expression was restricted to heart, skeletal muscle and PAECs of transgenic animals. Cultivated hA20-transgenic PAECs were protected against TNF-α-mediated apoptosis, and partially protected against CD95(Fas)L-mediated cell death; cardiomyocytes were partially protected in I/R. These findings reveal hA20 as a promising molecule for controlling AVR in multi-transgenic pigs for xenotransplantation studies. [ABSTRACT FROM AUTHOR]

Background: Despite major improvements in pig‐to‐primate xenotransplantation, long‐term survival of xenografts is still challenging. The major histocompatibility complex (MHC) class I, which is crucial in cellular immune response, is an important xenoantigen. Abrogating MHC class I expression on xenografts might be beneficial for extending graft survival beyond current limits. Methods: In this study, we employed the CRISPR/Cas9 system to target exon 2 of the porcine beta‐2‐microglobulin (B2M) gene to abrogate SLA class I expression on porcine cells. B2M‐KO cells served as donor cells for somatic cell nuclear transfer, and cloned embryos were transferred to three recipient sows. The offspring were genotyped for mutations at the B2M locus, and blood samples were analyzed via flow cytometry for the absence of SLA class I molecules. Results: Pregnancies were successfully established and led to the birth of seven viable piglets. Genomic sequencing proved that all piglets carried biallelic modifications at the B2M locus leading to a frameshift, a premature stop codon, and ultimately a functional knockout. However, survival times of these animals did not exceed 4 weeks due to unexpected disease processes. Conclusion: Here, we demonstrate the feasibility of generating SLA class I knockout pigs by targeting the porcine beta‐2‐microglobulin gene using the CRISPR/Cas9 system. Additionally, our findings indicate for the first time that this genetic modification might have a negative impact on the viability of the animals. These issues need to be solved to unveil the real value for xenotransplantation in the future. [ABSTRACT FROM AUTHOR]

Porcine induced pluripotent cells (iPSC) are considered an important large animal model for developing personalized stem cell therapies. Since the derivation of the first mouse and human iPSC, there have been relatively few reports regarding the reprogramming of pig somatic cells into pluripotency, exclusively with the use of transcription factors from human and mouse origin. To investigate whether using species-specific transcription factors would allow for an efficient reprogramming of porcine somatic cells, we have developed a Sleeping Beauty (SB) transposon system based on the porcine OCT4, SOX2, C-MYC, and KLF4 sequences and have tested it in the reprogramming of mouse and porcine fetal fibroblasts and pig EGC-like cells. Transfection of mouse embryonic fibroblasts with a multicistronic SB-tetO-pOCT4-F2A-pSOX2-T2A-pC-MYC-E2A-pKLF4-ires-Tomato vector resulted in the formation of iPSC colonies, which inactivated the exogenous transcription factors and upregulated their endogenous pluripotency genes. These cells maintained mESC-like morphology, formed embryoid bodies, and differentiated into different cell types in culture, including rhythmically contracting cardiac myocytes. In contrast, porcine fibroblasts and EGC-like cells transfected with the same transposon vector did not proliferate in culture and did not form any iPSC colonies. We then transfected these cells with multiple bi-cistronic vectors SB-Ef1a-pOCT4-ires-Tomato, SB-Ef1a-pSOX2-ires-Neo, SB-Ef1a-pC-MYC-ires-Puro, and SB-Ef1a-pKLF4-ires-Puro. As a result, both cell types formed multiple colonies with mouse ESC-like morphology. Clones established from individually picked colonies from transfected fetal fibroblasts maintained this morphology for 5-6 passages, after which they became flat and epithelial-like. They expressed endogenous SOX2, C-MYC, KLF4, and E-Cadherin, but not OCT4. At the same time, clones derived from EGC-like cells proliferated at accelerated rate and maintained their morphology for over 10 passages at the time of this writing. While the exogenous genes were expressed continuously during this period, the cells expressed also endogenous OCT4, SOX2, TERT, STELLA, TDH, and CHD1; however, C-MYC, KLF4, NANOG, and E-Cadherin expression was relatively low. These cells are currently being characterized for pluripotency. Despite the use of porcine transcription factors, the overall reprogramming of porcine cells appears to be still less efficient compared with mouse fibroblasts. Our results suggest that the species and tissue origin of the somatic cells may play a more important role in the reprogramming to pluripotency than the origin of the transcription factors used. In this respect, optimization of culture conditions may be necessary in order to allow for efficient and complete reprogramming of porcine somatic cells. This work is supported by a research grant by the Deutsche Forschungsgemeinschaft. [ABSTRACT FROM AUTHOR]

An abstract of the article "Recent progress in the production of transgenic pigs," by Heiner Niemann and colleagues is presented.