Use In Xenotransplantation Research Articles

Anatomical parameters do not determine linear breaking strength or dimensions of the porcine biliary system

Abstract Background The porcine gall bladder and cystic duct gained attention in experimental research aimed at improving human clinical care. While the common bile duct has been investigated before, there is almost no data on the porcine cystic duct. Its relevance for research originates from its potential use in xenotransplantation. Methods We included 10 consecutive pigs (1♀, 9♂) that had been sacrificed after participation in another study. At necropsy, the distance from the biliary papilla to the gall bladder (gall bladder distance) was measured. The cystic duct was then subjected to linear traction. Ethical approval for the study was granted. Associations between force measurements and anatomical parameters were tested by correlation analysis. Results The pigs had a mean body weight of 21.9 kg (standard deviation 2 kg), a mean liver weight of 590 g (standard deviation 88 g), and a mean crown rump length of 67.3 cm (standard deviation 3 cm). The gall bladder distance was 8.2 cm (95% confidence interval: 7.6–8.7 cm). The cystic duct withstood mean linear traction forces of 4.8 N (95% confidence interval: 3.7–5.8 N) and could be elongated by a mean of 6 mm (95% confidence interval: 3.9–8 mm). Linear breaking strength was neither correlated to gall bladder distance (R = 0.3, 95% confidence interval: − 0.41 to 0.78, P = 0.406) or crown rump length (R = 0.42, 95% confidence interval: − 0.28 to 0.83, P = 0.222) nor liver weight (R = 0.02, 95% confidence interval: − 0.62 to 0.64, P = 0.954) or body weight (R = 0.36, 95% confidence interval: − 0.35 to 0.81, P = 0.304). This was also true for gall bladder distance. Conclusions Our study indicates that allometric parameters were not associated with the gall bladder distance or its resistance to traction forces. Consequently, substantial variation of these parameters can be expected in these surgically important parameters, which cannot be derived from easily accessible anatomical parameters.

Atrase B, a novel metalloprotease with anti-complement and anti-coagulant activity, significantly delays discordant cardiac xenograft rejection.

Both the complement and the coagulation systems play important roles in the development of hyperacute or acute antibody-mediated xenograft rejection. Atrase B is a novel metalloproteinase isolated from the venom of Naja atra. In this study, we investigated the inhibitory effects of atrase B on complement activation and coagulation, as well as the effect on xenograft survival in a discordant xenotransplantation model. The in vitro anti-complement activity of atrase B was evaluated using a normal human serum (NHS)-mediated complement-dependent cytotoxicity model with an immortalized porcine aortic endothelial cell line (iPEC) as the target. The in vivo inhibitory effects on complement activity and coagulation function were measured in rats after the administration of atrase B. Guinea pig hearts were transplanted heterotopically into Wistar rats with or without atrase B pre-treatment. Pre-treatment of the NHS with atrase B inhibited the cell lysis of iPECs in a dose-dependent manner. FACS analysis showed that atrase B potently suppressed the deposition of C5b-9, but not C3c and C4c, on iPECs. In vivo, atrase B-treated rats showed a significant reduction in serum complement activity; markedly prolonged PT, APTT, and TT; and a decreased plasma level of fibrinogen. When compared to PBS treatment evaluated at study endpoint, atrase B treatment significantly delayed xenograft rejection and attenuated pathologic damage, the formation of platelet microthrombi, and the deposition of fibrin and C5b-9. The dual activities of anti-complement and anti-coagulation make atrase B a potential adjuvant therapeutic drug for use in xenotransplantation.

FEATURES OF THE IMMUNOGENETIC STRUCTURE OF PIGS OF DOMESTIC BREEDS, SUITABLE FOR XENOTRANSPLANTATION

The aim of the work was to determine the immunogenetic characteristics of pigs of two Ukrainian breeds by their adaptability, resistance, reproduction, genetic homogeneity, the presence of alleles that determine the potential suitability for use in xenotransplantation and other biomedical purposes. At present, there is no breed or specialized line of pigs in Ukraine to address the urgent problems of humane medicine. However, there is a favorable situation for the creation of such a breed or the withdrawal of a specialized line because of the demand of pharmacological concerns for model biological objects to study the mechanisms of action of modern medical preparations, to develop methods of conducting bloodless surgical operations, modeling of the clinic and epidemiology of infectious diseases of different diseases. immune response, etc. In this context, there was a compelling reason to preserve the indigenous breeds of pigs of Ukraine, especially Ukrainian meat and Мyrgorod. Standard methods of immunogenetic analysis of pigs were used. The erythrocyte antigens of 9 blood group systems were determined using specific immune sera and the involvement of a bank of immunodiagnostics meeting international requirements. Blood groups were determined by the reaction of agglutination, an indirect Coombs test, and a hemolytic test. Statistical processing of the research results was performed using mathematical statistics using the GenAlex-6.0 computer program. As a result of the immunogenetic analysis of Ukrainian beef and myrrh-pig breeds, it was determined that each of them is characterized by a specific immunogenic profile, which is related to both breed characteristics and differences, and methods of their breeding. The difference between the distribution of the overwhelming number of alleles of blood groups was statistically significant. Pigs of Ukrainian meat and Мyrgorod breeds had the largest differences in allele distribution by B, E, F, K, L blood group systems with the presence of the Ladhjk marker allele in the latter (p < 0.05). The specificity of immunogenic profiles was evaluated, depending on the history of creation and the direction of the animal's performance. Species of Myrhorod and Ukrainian meat breeds with the presence of genotypes A - / - and Ebdgkmp / bdgkmp were found to determine the suitability of animals for xenotransplantation. Of the 80 animals tested for immunogenetic markers, only 24 individuals met the established selection criteria. The range of determined indicators of actual homozygosity for selected genotype animals ranged from 55.56% to the maximum value – 88.89%, mainly in sows from the Smorodinа, Rusalkа, Sorokа and Soyka families. According to molecular genetic analysis, you have been informed that the established criteria of the biomedical model meet only 13 individuals of Ukrainian meat breed. Among animals with the highest index of actual homozygosity, representatives of Cererа and Cіlina were identified, with Celina 4092 being homozygous for all blood group systems studied. The selection of pigs for blood alleles, desirable for xenotransplantation, with their transfer to the homozygous state: A -/- and Ebdgkmp / bdgkmp will impair the adaptive, first of all, reproductive qualities of the animals and create significant problems for the cultivation of such individuals. The possibility of the occurrence of reproductive and resistance disorders of pigs in the selection of blood groups genotypes modeled for xenotransplantation and the search for ways to overcome them are being considered.

Optimizing sgRNA length to improve target specificity and efficiency for the GGTA1 gene using the CRISPR/Cas9 gene editing system.

The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production.

325 PRODUCTION OF GAL KNOCKOUT/hA20 TRANSGENIC PIGS WITH IMPROVED XENOPROTECTIVE PROPERTIES

Pig-to-human xenotransplantation is promising for overcoming the shortage of suitable human donor organs, but is hampered by immunological barriers. The next immunological hurdle is the acute vascular rejection (AVR), which is associated with activation of the endothelium and the coagulation system. Recently, we demonstrated that transgenic expression of the zinc finger protein A20 protects porcine cells against apoptotic and inflammatory stimuli (Oropeza et al. 2009 Xenotransplantation 16, 522–534). Compared with other anti-apoptotic proteins, A20 also has immune-modulatory potential as shown in an CD95(Fas)Ligand assay. However, in that study, hA20 was only expressed in skeletal muscle, heart and porcine aortic endothelial cells of transgenic pigs. For use in xenotransplantation, it is critical to produce pigs with ubiquitous expression of hA20. Here, we constructed a new vector based on the Sleeping Beauty transposon plasmid pT2/HB containing the hA20 cDNA driven by the ubiquitously and strongly expressing CAGGS-promoter and co-transfected gal–/– porcine fibroblasts (Hauschild et al. 2011 Proc. Natl. Acad. Sci. USA 108, 15 010) together with the SB transposase 100X plasmid (pT2/HB and SB transposase both kindly provided by Dr. Zoltan Ivics). Cells were selected with 400 µg of G418/mL of medium for 14 days. Subsequently, cells were screened by PCR. Transfected cell clones were pooled and used as donor cells in somatic cell nuclear transfer. Reconstructed embryos were transferred to 2 synchronized sows. Both remained pregnant on Day 25 of gestation. One recipient is expected to deliver in September 2012. The second sow, which received 104 embryos, was sacrificed on Day 26 of pregnancy and 2 fetuses could be obtained (cloning efficiency 1.92%). The fetuses had integrated the transgene into their genome as shown by PCR. Real-time PCR results from fetal fibroblasts indicated similar hA20 mRNA expression levels in both fetuses, whereas wild type controls were negative. The hA20 expression level was 2.5 and 3.1 times lower than in the original pooled of transfected cells. Fetus #1, which showed a slightly higher hA20 mRNA expression, was used for recloning. In total, 3 more recipients received an average of 104 hA20 transgenic embryos each. Currently, the hA20 protein level in fetal fibroblasts is determined by fluorescence activated cell sorting analysis. Once pigs are born, the tissue distribution of hA20 will be analysed. In parallel, the function of the transgene will be studied in the CD95(Fas)Ligand assay. Hearts and kidneys of the hA20-transgenic pigs will be further tested in ex vivo perfusion assays and in a pig-to-baboon xenotransplantation. This approach is promising for advancing pig-to-human xenotransplantation to preclinical application.

GalTKO.hCD46 Transgenic Pig Lungs: 4-Year Experience in a Xenogenic Perfusion Model

Purpose: Perfusion of wild type pig lungs with human blood leads within minutes to hyperacute rejection that is associated with the immediate sequestration of circulating platelets and neutrophils and the activation of the coagulation and complement cascade. The generation of GalTKO pigs has significantly improved survival, however, the described reactions are still present even though at a lower level only. To further control the activation of the complement cascade, a human complement regulatory protein (hCPRP) gene was inserted on the basis of a GalTKO-pig. Those GalTKO.hCD46 transgenic organs have been use in xenotransplantation for several years now. Here we present the results of our 4-year experience with the use of GalTKO.hCD46 pig lungs in a xenogenic lung perfusion model. Methods: Wildtype (n=17), GalTKO (n=16) and GalTKO.hCD46 transgenic pig lungs (n=31) were perfused with heparinized fresh human blood until failure or elective termination after 4 hours. At prespecified time points, blood and tissue samples were collected. Pulmonary artery (PA) flow and pressure were recorded online. Results: Median survival time (MST) in the wild type group was 10‘, in the GalTKO cohort 120‘. The additional expression of hCD46 showed a trend to higher MST of 180′ (p=0.21). Pulmonary vascular resistance was not improved in the GalTKO.hCD46 group when compared to GalTKO alone. Platelet sequestration was significantly reduced in the early perfusion phase (% initial platelets remaining at 5‘: GalTKO.hCD46 51±34 vs. GalTKO 26±14, p=< 0.05) but not at the later time points. Complement activation measured by C3a level was lower at all time points in the GalTKO.hCD46 group with statistically highly significant lower level after 2h of perfusion (GalTKO.hCD46 237±175 vs. GalTKO 873±1002, p= 0.009). Conclusion: Complement activation is better controlled in GalTKO pig lungs genetically modified to express hCD46, and presumably contributes to the trend toward longer survival of those organs. However, various other rejection mechanisms like activation of the coagulation cascade, platelet sequestration, and/or non-Gal antibody-mediated rejection lead to organ injury. Additional interventions targeted to these mechanisms will very likely be necessary to further advance lung xenotransplantation toward clinical application.

Successful Suppression of Endogenous &lt;i&gt;&amp;alpha;-1,3-Galactosyltransferase&lt;/i&gt; Expression by RNA Interference in Pig Embryos Generated &lt;i&gt;In Vitro&lt;/i&gt;

RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B(4), a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of double-stranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation.

Porcine pathogens and xenotransplantation: PERV and beyond!

Concerns regarding the transmission of potentially zoonotic porcine viruses via a xenotransplant have prompted a significant number of studies on methods to eliminate or prevent expression and transmission of these viruses. The main focus of these studies, to date, has been the porcine endogenous retrovirus (PERV); PERV is a genetically acquired element and present in the genome of all swine. This situation is problematic as it cannot simply be eliminated from swine by using methods currently employed to exclude exogenous pathogens in barrier facilities. As such, alternative strategies have been sought to circumvent the potential risk of PERV expression and transmission via a xenotransplant, however, there are other existing and emerging pathogens of concern that should be addressed when using this novel technology in vivo.Zoonotic porcine viruses have been identified that require specific diagnostic methods to confirm their absence. Animal husbandry and the exclusion of pathogens from SPF herds for use in xenotransplantation have been widely discussed and a number of organizations have issued guidelines on the screening for infectious agents. Although these recommendations on monitoring protocol and the identification of adventitious agents are clear, there is no comprehensive list of pathogens to be excluded from these animals that can be applied to all centres carrying out xenotransplantation. Currently, SPF animals used for research purposes are monitored for specific pathogens as defined by local guidelines, and may not be tested for all pathogens relevant to xenotransplantation.As recent data has indicated the potential for certain porcine pathogens to cross the species barrier, it is clear that xenotransplantation is a unique situation which may require us to address a more comprehensive panel of microorganisms than is currently recommended for SPF animals. This presentation will discuss data on the presence of pathogens in pigs, other than PERV, that may cause concern during the clinical application of xenotransplantation and the issues regarding the potential transfer of new zoonotic microorganisms.

T regulatory cells in xenotransplantation

The role of T regulatory cells (Treg) in the induction and maintenance of allograft tolerance is being studied to a great extent. In contrast, little is known on their potential to prevent graft rejection in the field of xenotransplantation, where acute vascular rejection mediated by cellular and humoral mechanisms and thrombotic microangiopathy still prevents long-term graft survival. In this regard, the induction of donor-specific tolerance through isolation and expansion of xenoantigen-specific recipient Treg is currently becoming a focus of interest. This review will summarize the present knowledge concerning Treg and their potential use in xenotransplantation describing in particular CD4(+)CD25(+)Foxp3(+) T cells, CD8(+)CD28(-) Treg, double negative CD4(-)CD8(-) T cells, and natural killer Treg. Although only studied in vitro so far, human CD4(+)CD25(+)Foxp3(+) Treg is currently the best characterized subpopulation of regulatory cells in xenotransplantation. CD8(+)CD28(-) Treg and double negative CD4(-)CD8(-) Treg also seem to be implicated in tolerance maintenance of xenografts. Finally, one study revealing a role for natural killer CD4(+)Valpha14(+) Treg in the prolongation of xenograft survival needs further confirmation. To our opinion, CD4(+)CD25(+)Foxp3(+) Treg are a promising candidate to protect xenografts. In contrast to cadaveric allotransplantation, the donor is known prior to xenotransplantation. This advantage allows the expansion of recipient Treg in a xenoantigen specific manner before transplantation.

Distribution and expression of porcine endogenous retroviruses in multi‐transgenic pigs generated for xenotransplantation

Multi-transgenic pigs produced for use in xenotransplantation have to be screened for the presence and expression of porcine endogenous retroviruses (PERV) to select animals with low PERV load. The production of transgenic pigs may also be associated with the integration of the transgene adjacent to or into the locus of a PERV provirus, potentially leading to an enhanced virus expression. Non-transgenic animals, single-transgenic, and multi-transgenic pigs were screened for the presence of PERV-A, -B, and -C and recombinant PERV-A/C using polymerase chain reaction (PCR). PERV expression was determined by real time reverse transcriptase-PCR. An assay based on the activation of PERV in peripheral blood mononuclear cells by mitogens was used to discriminate between low and high PERV producer animals. All animals carried PERV-A and -B. A total of 176 from 181 (97.2%) animals carried PERV-C in the germ line and 18 from 64 animals carried PERV-A/C in the genome of lymphoid cells but not in the germ line. The expression of PERV was very low in all animals and not different between transgenic pigs and non-transgenic animals. PERV expression differed between various pig lines. The highest expression was found in mini-pigs and crossing other pig lines with mini-pigs resulted in increased PERV expression in the progeny. However, expression of viral proteins and particle release were not observed in all transgenic animals. No evidence for elevated PERV expression in (multi-) transgenic pigs was observed. Differences in PERV expression correlated with the genetic background of the animals, not with the specific transgene. Mini-pigs consistently had the highest level of PERV expression and animals with a mini-pig background had a higher level of expression compared with animals without mini-pig background.

Concise classification of the genomic porcine endogenous retroviral γ1 load to defined lineages

We investigated the infection history of porcine endogenous retroviruses (PERV) γ1 by analyzing published env and LTR sequences. PERV sequences from various breeds, porcine cell lines and infected human primary cells were included in the study. We identified a considerable number of retroviral lineages indicating multiple independent colonization events of the porcine genome. A recent boost of the proviral load in an isolated pig herd and exclusive occurrence of distinct lineages in single studies indicated the ongoing colonization of the porcine genome with endogenous retroviruses. Retroviral recombination between co-packaged genomes was a general factor for PERV γ1 diversity which indicated the simultaneous expression of different proviral loci over a period of time. In total, our detailed description of endogenous retroviral lineages is the prerequisite for breeding approaches to minimize the infectious potential of porcine tissues for the subsequent use in xenotransplantation.

Play it in E or G: utilization of HLA‐E and ‐G in xenotransplantation

Human NK cell-mediated graft rejection is likely to be one of several biological obstacles to routine pig-to-human xenotransplantation. Abrogating NK cell activation by either elimination of activating ligands on porcine cells or expression of molecules serving as ligands for NK cell inhibitory receptors, or both, could overcome this hurdle. HLA-E and -G exhibit very limited polymorphism and are ligands for NK cell inhibitory receptors. This review summarizes successes and limitations of their use in xenotransplantation as inferred from ex vivo analyses of NK cell activity, highlights potential effects they may have on T-cell responses, and considers prospects of preclinical trials and potential outcomes.

Human amniotic fluid-derived stem cells are rejected after transplantation in the myocardium of normal, ischemic, immuno-suppressed or immuno-deficient rat

Human amniotic fluid-derived stem (AFS) cells, similarly to embryonic stem cells, could possess privileged immunological characteristics suitable for a successful transplantation even in a discordant xenograft system. We investigated whether AFS cells could be fruitfully used in a rat model of myocardial infarction. c-kit immunomagnetic-sorted AFS cells were characterized by flow cytometric analysis and cytospins as well as reverse-transcription polymerase chain reaction, Western blotting and immunocytochemistry for cardiovascular differentiation markers. In vitro, AFS cell phenotypic conversion was assayed by cardiovascular-specific induction media or co-cultured with rat neonatal cardiomyocytes. AFS cells showed mRNAs and/or protein for endothelial (angiopoietin, CD146) and smooth muscle (smoothelin) cells, and cardiomyocyte (Nkx2.5, MLC-2v, GATA-4, β-MyHC) markers. Acquisition of a cardiomyocyte-like phenotype in rare AFS cells could be seen only in co-cultures with rat neonatal cells. In vivo, AFS cells xenotransplantated in a rat model of myocardial infarction, with or without cyclosporine treatment, or in intact heart from immuno-competent or immuno-deficient animals were acutely rejected due to the different recruitment of recipient CD4 +, CD8 + T and B lymphocytes, NK cells and macrophages. This reaction is most likely to be linked to expression of B7 co-stimulatory molecules CD80 and CD86 as well as macrophage marker CD68 on AFS cells. Xenotransplanted AFS cells gave also rise in some animals to cell masses in the subendocardium and myocardium suggestive of a process of chondro-osteogenic differentiation. Despite AFS cells in vitro can differentiate to some extent to cells of cardiovascular lineages, their in vivo use in xenotransplantation for cell therapy of myocardial infarction is hampered by their peculiar immunogenic properties and phenotypic instability.

Genetic and functional evaluation of the level of inbreeding of the Westran pig: a herd with potential for use in xenotransplantation

The Westran pig has been purposely inbred for use in xenotransplantation. The herd originated in the wild from a limited gene pool and has been inbred by repeated full-sib matings for nine generations. The aim of this study was to evaluate the level of inbreeding by functional assays, such as bi-directional MLR and reciprocal skin grafts between herd members, and by genetic analysis using highly polymorphic genetic markers to calculate the level of inbreeding. The MLR between herd members were non-reactive whereas there was a prompt response to third party pig lymphocytes, indicative of a normal immune responsiveness in Westran pigs but isogenicity of the major histocompatibility complex. Skin grafts between male siblings or female sibling skin grafts on male recipients showed prolonged survival but with few exceptions did not survive beyond 100 days suggesting that by the fifth generation the Westran herd was still mismatched at minor histocompatibility antigens. This level of functional inbreeding was confirmed by microsatellite analysis of highly polymorphic markers, which showed that 52 of 53 chromosomally dispersed markers were fixed by the ninth generation. This level of fixation was consistent with 19 to 20 generations of full-sibling inbreeding. The calculated inbreeding coefficient at generation 10 was 0.98159. This analysis confirms that the Westran pig is highly inbred and we propose that analysis of chromosomally dispersed highly polymorphic markers is an accurate and reproducible method for assessing the level of inbreeding of a pig herd.

Novel herpesviruses of Suidae: indicators for a second genogroup of artiodactyl gammaherpesviruses.

Five novel herpesviruses were identified in suid species from Africa (common warthog, Phacochoerus africanus) and South-East Asia (bearded pig, Sus barbatus; babirusa, Babyrousa babyrussa) by detection and analysis of their DNA polymerase genes. Three of the novel species, P. africanus cytomegalovirus 1, P. africanus lymphotropic herpesvirus 1 (PafrLHV-1) and S. barbatus lymphotropic herpesvirus 1 (SbarLHV-1), were closely related to known beta- (porcine cytomegalovirus) and gammaherpesviruses [porcine lymphotropic herpesvirus (PLHV) 1 and 3] of domestic pigs. In contrast, two novel species, S. barbatus rhadinovirus 1 (SbarRHV-1) and Babyrousa babyrussa rhadinovirus 1 (BbabRHV-1), were more closely related to a ruminant gammaherpesvirus, bovine herpesvirus 4 (BoHV-4), than to the porcine gammaherpesviruses PLHV-1, -2, -3, PafrLHV-1 and SbarLHV-1. SbarRHV-1, BbabRHV-1 and BoHV-4 were therefore tentatively assigned to a novel genogroup of artiodactyl gammaherpesviruses. This latter genogroup may also contain an as yet undiscovered gammaherpesvirus of domestic pigs, thereby adding a concern to their use in xenotransplantation.

Monitoring for potentially xenozoonotic viruses in New Zealand pigs.

Shortage of human donor organs for transplantation has prompted evaluation of animals as an alternative donor source. Pigs are the most acceptable candidate animals but issues of xenozoonozes remain. Despite careful monitoring of high-health-status (HHS) pigs, there is still a risk that their tissues may carry infectious agents. Furthermore, pathogens which are significant in xenotransplantation are not necessarily those of veterinary importance. The detection of these potentially transmissible infectious agents may require the development and application of new surveillance technologies. We present data on monitoring for five potentially xenotic viruses in New Zealand pig herds, namely pig cytomegalovirus (PCMV), pig lymphotropic herpesvirus (PLHV), encephalomyocarditis virus (EMCV), pigcircovirus (PCV), and hepatitis E virus (HEV). These five viruses are either potentially oncogenic, establish persistent infection, or are known to be zoonotic. This study has expanded significantly the information on porcine viruses in New Zealand. Using this information, it is now possible to complete protocols for monitoring pig herds and tissues prior to their use in xenotransplantation. The study resulted in selection of a possible source herd for swine-to-human islet transplantation.

Production of cloned pigs by using somatic cells as donors.

NUCLEAR TRANSFER is a procedure by which genetically identical individuals can be created. The applications of these techniques for nuclear transfer will be in agricultural, biomedical and basic research. Based on the sources of donor cells, nuclear transfer can be classified into embryonic cell nuclear transfer and somatic cell nuclear transfer. Offspring from cultured cells were first reported in 1994 (Sims and First, 1994) in cattle. Since this paper showed that cells could be cultured at least 28 days prior to nuclear transfer, it held great promise for genetically modifying the cells prior to nuclear transfer, and thus the possibility of producing animals with specific genetic modifications. This concept is exemplified by the generation of transgenic sheep (Schnieke et al., 1997), pigs (Park et al., 2001), calves (Cibelli et al., 1998a), and gene-targeted sheep (McCreath et al., 2000) and pigs (Lai et al., 2002b), derived from nuclear transfer approaches by using transfected somatic cells. For pigs, somatic cell nuclear transfer has another specific significance, as is it would allow the use of genetic modification procedures to produce tissues and organs from cloned pigs with reduced immunogenicity for use in xenotransplantation (Lai et al., 2002b). However, the efficiency of somatic cell nuclear transfer, when measured as development to term as a proportion of oocytes used, has been very low (1–2%). A number of variables influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to nuclear transfer, the method of artificial oocyte activation, embryo culture, possible loss of somatic imprinting in the nuclei of reconstructed embryos, failure of adequate reprogramming of the transplanted nucleus, and the techniques employed for nuclear transfer. In pig, there are additional difficulties in that the quality of embryos produced in vitro is low, and at least four good embryos are required to initiate and establish a pregnancy. Procedures for nuclear transfer include the following steps: acquisition of recipient oocytes and donor cells, enucleation (removal of the chromosome from recipient oocytes), insertion of donor nuclei into enucleated oocytes, artificial activation of reconstructed oocytes, and embryo transfer (transfer of the reconstructed embryos into a surrogate).

Production of cloned pigs from in vitro systems.

Here we describe a procedure for cloning pigs by the use of in vitro culture systems. Four healthy male piglets from two litters were born following nuclear transfer of cultured somatic cells and subsequent embryo transfer. The initiation of five additional pregnancies demonstrates the reproducibility of this procedure. Its important features include extended in vitro culture of fetal cells preceding nuclear transfer, as well as in vitro maturation and activation of oocytes and in vitro embryo culture. The cell culture and nuclear transfer techniques described here should allow the use of genetic modification procedures to produce tissues and organs from cloned pigs with reduced immunogenicity for use in xenotransplantation.

Genetic characterization of type 2 porcine circovirus (PCV-2) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of North America and development of a differential PCR-restriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1 and PCV-2.

Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine. Increasing evidence indicates that a variant strain of porcine circovirus (PCV), designated type 2 PCV (PCV-2), is responsible for PMWS. To determine the extent of genetic heterogeneity of PCV-2 isolates, the complete genomes of six PCV-2 isolates from different regions of North America were amplified by PCR and sequenced. Sequence and phylogenetic analyses confirmed that two distinct genotypes of PCV exist: nonpathogenic genotype PCV-1 and PMWS-associated genotype PCV-2. However, within the PCV-2 genotype, several minor branches that have been identified appear to be associated with geographic origins. The genomic sequences of two French PCV-2 isolates diverge the most from those of other PCV-2 isolates and form a distinct branch. Other minor but distinguishable branches have also been identified for a Taiwan PCV-2 isolate and two of the Canadian PCV-2 isolates. All the U.S. PCV-2 isolates are closely related, but the Canadian isolates vary, to some extent, in their genomic sequences. The data from this study indicate that although the genome of PCV-2 is generally stable among different isolates, PCV-2 isolates from different geographic regions vary in their genomic sequences. This variation may have important implications for PCV-2 diagnosis and research. On the basis of genetic analyses of available PCV strains, a universal PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed to detect and differentiate between infections with PCV-1 and PCV-2. This PCR-RFLP assay should be useful for studying the pathogenesis of PCV-2, for detecting PCV-2 infection in pigs from different geographic regions, and for screening donor pigs for use in xenotransplantation.

Targeted modification of the domestic animal genome

While the technique of homologous recombination, or gene targeting, has led to the generation of transgenic mice of great value to biomedical research, similar approaches are only being developed in other species. With the exception of recent reports on the generation of genetargeted sheep, the technology in domestic animals is still in its infancy (45). The development of techniques for generating large animals with deleted or modified genes will result in the generation of animals of great value to society. While the technical difficulties to achieve gene targeting in domestic species are significant, they are not insurmountable. Potential applications in both the bovine and porcine species are described with particular emphasis on the generation of cattle resistant to bovine spongiform encephalopathy (BSE) and pigs that can be of use in xenotransplantation.