Transgenic Pigs Research Articles

A pilot study of diabetic retinopathy in a porcine model of maturity onset diabetes of the young type 3 (MODY3)

Diabetic retinopathy (DR) is a leading cause of blindness in the working population. Because novel therapeutic intervention require testing, there is an urgent need for reliable animal models that faithfully replicate DR. Pig eyes have many similarities to human eyes anatomically and physiologically. Thus, attempts have been made to establish porcine models of DR by surgical, pharmaceutical or genetical induction of insulin deficiency, and dietary intervention. A previous study reported a transgenic pig model of maturity onset diabetes of the young type 3 (MODY3) developed signs of severe DR such as hemorrhage and proliferative tissue at the surface of the retina. However, the course of development of DR has not been studied in detail in this model. The purpose of this study was to investigate the early phase of DR in a MODY3. MODY3 and wild-type (WT) pigs underwent fundus photography and fluorescein angiogram (FA) before they developed cataracts. Animals were euthanized at age 1, 4, 7, and 10 months. Whole-mount retina and 10-μm thick paraffinized sections were stained with isolectin B4, and vessel density was determined by MATLAB software. At 4 and 7 months, retinal arterioles were immediately cannulated, and vasomotor action was measured by incubation with bradykinin and sodium nitroprusside. In the MODY3 pigs, fasting blood sugar levels gradually increased up to 500 mg/dL. Vascular tortuosity and yellowish spindle-shaped lesions were confirmed in MODY3 pigs at the age of 7 months; however, no microaneurysms were detected on FA. Compared with age-matched WT pigs, MODY3 pigs showed a significant decrease in blood vessel density in the intermediate and deep vascular plexus at 4 and 7 months of age and a slight decrease in capillary density in the superficial vascular plexus at 7 months of age. In MODY3 pigs, electron microscopy revealed thickening of the capillary basement membrane and leukostasis in the major blood vessels at 10 months of age. Bradykinin-induced dilation of retinal arterioles was diminished in MODY3 pigs as early as 7 months of age. Within 1 year after birth, MODY3 pigs show all typical early vascular lesions of diabetes except for microaneurysm formation. This pilot study suggests that the MODY3 pigs may serve as a suitable DR model to test effects of newly developed compounds on DR.

In defense of xenotransplantation research: Because of, not in spite of, animal welfare concerns.

It is envisioned that one day xenotransplantation will bring about a future where transplantable organs can be safely and efficiently grown in transgenic pigs to help meet the global organ shortage. While recent advances have brought this future closer, worries remain about whether it will be beneficial overall. The unique challenges and risks posed to humans that arise from transplanting across the species barrier, in addition to the costs borne by non-human animals, has led some to question the value of xenotransplantation altogether. In response, we defend the value of xenotransplantation research, because it can satisfy stringent welfare conditions on the permissibility of animal research and use. Along the way, we respond to the alleged concerns, and conclude that they do not currently warrant a cessation or a curtailing of xenotransplantation research.

Auxiliary liver xenotransplantation technique in a transgenic pig to non-human primate model: a surgical approach to prolong survival and better understand graft rejection

Auxiliary liver xenotransplantation technique in a transgenic pig to non-human primate model: a surgical approach to prolong survival and better understand graft rejection

Establishment of transgenic pigs overexpressing human PKD2-D511V mutant.

Numerous missense mutations have been reported in autosomal dominant polycystic kidney disease which is one of the most common renal genetic disorders. The underlying mechanism for cystogenesis is still elusive, partly due to the lack of suitable animal models. Currently, we tried to establish a porcine transgenic model overexpressing human PKD2-D511V (hPKD2-D511V), which is a dominant-negative mutation in the vertebrate in vitro models. A total of six cloned pigs were finally obtained using somatic cell nuclear transfer. However, five with functional hPKD2-D511V died shortly after birth, leaving only one with the dysfunctional transgenic event to survive. Compared with the WT pigs, the demised transgenic pigs had elevated levels of hPKD2 expression at the mRNA and protein levels. Additionally, no renal malformation was observed, indicating that hPKD2-D511V did not alter normal kidney development. RNA-seq analysis also revealed that several ADPKD-related pathways were disturbed when overexpressing hPKD2-D511V. Therefore, our study implies that hPKD2-D511V may be lethal due to the dominant-negative effect. Hence, to dissect how PKD2-D511V drives renal cystogenesis, it is better to choose in vitro or invertebrate models.

Application of the transgenic pig model in biomedical research: A review

The large animal model has gradually become an essential part of preclinical research studies, relating to exploring the disease pathological mechanism, genic function, pharmacy, and other subjects. Although the mouse model has already been widely accepted in clinical experiments, the need for finding an animal model with high similarity compared with a human model is urgent due to the different body functions and systems between mice and humans. The pig is an optimal choice for replacement. Therefore, enhancing the production of pigs used for models is an important part of the large animal model as well. Transgenic pigs show superiority in pig model creation because of the progress in genetic engineering. Successful cases of transgenic pig models occur in the clinical field of metabolic diseases, neurodegenerative diseases, and genetic diseases. In addition, the choice of pig breed influences the effort and efficiency of reproduction, and the mini pig has relative obvious advantages in pig model production. Indeed, pig models in these diseases provide great value in studies of their causes and treatments, especially at the genetic level. This review briefly outlines the method used to create transgenic pigs and species of producing transgenic pigs and provides an overview of their applications on different diseases and limitations for present pig model developments.

Xenotransplantation—reflections on the bioethics

AbstractBackgroundSimilar to most countries in the world, China has a severe shortage of human organs, and this is one of the main issues restricting the application of organ transplantation technology. In 2019 alone, only 19,454 (23.90%) of the 81,410 people waiting were able to receive organ transplants. There is an increasing focus from both the medical profession and society on how to fill the gap between supply and demand.MethodsXenotransplantation using animal organs is being considered as one option to make up for the shortage of human organs for transplantation. For some years now, the international medical community has been examining the possibility of using animal organs for human transplant. However, the research has faced two important types of challenges: scientific and ethical issues.ResultsIn January 2021, the first clinical trial of transgenic pig heart transplantation into a human recipient was completed by the Medical Center of the University of Maryland in the United States. This has stimulated enthusiasm and interest in xenotransplantation.ConclusionsThe trend towards xenotransplantation has highlighted global problems such as the severe shortage of organ transplant donors and the high cost of organ transplantation. China needs to consider how to cope with the scientific, public health, and social ethics challenges of xenotransplantation clinical trials.

Genetically Engineered Pigs as Efficient Salivary Gland Bioreactors for Production of Therapeutically Valuable Human Nerve Growth Factor.

Farm animal salivary glands hold great potential as efficient bioreactors for production of human therapeutic proteins. Nerve growth factor (NGF) is naturally expressed in animal salivary glands and has been approved for human clinical treatment. This study aims to employ transgenic (TG) pig salivary gland as bioreactors for efficient synthesis of human NGF (hNGF). hNGF-TG pigs were generated by cloning in combination with piggyBac transposon-mediated gene transfer. These hNGF-TG pigs specifically expressed hNGF protein in their salivary glands and secreted it at high levels into saliva. Surgical and nonsurgical approaches were developed to efficiently collect saliva from hNGF-TG pigs. hNGF protein was successfully purified from collected saliva and was verified to be biologically active. In an additional step, the double-transgenic pigs, where the endogenous porcine NGF (pNGF) gene was replaced by another copy of hNGF transgene, were created by cloning combined with CRISPR/Cas9-mediated homologous recombination. These double-transgenic pigs expressed hNGF but not pNGF, thus avoiding possible “contamination” of hNGF with pNGF protein during purification. In conclusion, TG pig salivary glands can be used as robust bioreactors for a large-scale synthesis of functional hNGF or other valuable proteins. This new animal pharming method will benefit both human health and biomedicine.

A bioartificial transgenic porcine whole liver expressing human proteins alleviates acute liver failure in pigs

BackgroundPreventing heterologous protein influx in patients is important when using xenogeneic bioartificial livers (BALs) to treat liver failure. The development of transgenic porcine livers synthesizing human proteins is a promising approach in this regard. Here, we evaluated the safety and efficacy of a transgenic porcine liver synthesizing human albumin (hALB) and coagulation factor VII (hFVII) within a bioartificial system. MethodsTibetan miniature pigs were randomly subjected to different interventions after surgery-induced partially ischemic liver failure. Group A (n = 4) was subjected to basic treatment; group B (n = 4) was to standard medical treatment and wild-type porcine BAL perfusion, and group C (n = 2) was to standard medical treatment and transgenic BAL perfusion. Biochemical parameters, coagulation status, survival time, and pathological changes were determined. Expressions of hALB and hFVII were detected using immunohistochemistry and enzyme-linked immunosorbent assays. ResultsThe survival time in group A was 9.75 ± 1.26 days; this was shorter than that in both perfused groups, in which all animals reached an endpoint of 12 days (P = 0.006). Ammonia, bilirubin, and lactate levels were significantly decreased, whereas albumin and fibrinogen levels were increased after perfusion (all P < 0.05). hALB and hFVII were detected in transgenic BAL-perfused pig serum and ex vivo in the liver tissues. ConclusionsThe humanized transgenic pig livers could synthesize and secrete hALB and hFVII ex vivo in a whole organ-based bioartificial system, while maintaining their metabolism, detoxification, transformation, and excretion functions, which were comparable to those observed in wild-type porcine livers. Therefore, the use of transgenic bioartificial whole livers is expected to become a new approach in treating acute liver failure.

Exogenous Melatonin in the Culture Medium Does Not Affect the Development of In Vivo-Derived Pig Embryos but Substantially Improves the Quality of In Vitro-Produced Embryos.

Cloned and transgenic pigs are relevant human disease models and serve as potential donors for regenerative medicine and xenotransplantation. These technologies demand oocytes and embryos of good quality. However, the current protocols for in vitro production (IVP) of pig embryos give reduced blastocyst efficiency and embryo quality compared to in vivo controls. This is likely due to culture conditions jeopardizing embryonic homeostasis including the effect of reactive oxygen species (ROS) influence. In this study, the antioxidant melatonin (1 nM) in the maturation medium, fertilization medium, or both media was ineffective in enhancing fertilization or embryonic development parameters of in vitro fertilized oocytes. Supplementation of melatonin in the fertilization medium also had no effect on sperm function. In contrast, the addition of melatonin to the embryo culture medium accelerated the timing of embryonic development and increased the percentages of cleaved embryos and presumed zygotes that developed to the blastocyst stage. Furthermore, it increased the number of inner mass cells and the inner mass cell/total cell number ratio per blastocyst while increasing intracellular glutathione and reducing ROS and DNA damage levels in embryos. Contrarily, the addition of melatonin to the embryo culture medium had no evident effect on in vivo-derived embryos, including the developmental capacity and the quality of in vivo-derived 4-cell embryos or the percentage of genome-edited in vivo-derived zygotes achieving the blastocyst stage. In conclusion, exogenous melatonin in the embryo culture medium enhances the development and quality of in vitro-derived embryos but not in in vivo-derived embryos. Exogenous melatonin is thus recommended during embryo culture of oocytes matured and fertilized in vitro for improving porcine IVP efficiency.

A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model

Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological rejection, which is the main hurdle in pig-to-human xenotransplantation, various engineered transgenic pigs have been developed. Ablation of xeno-reactive antigens, especially the 1,3-Gal epitope (GalT), which causes hyperacute rejection, and insertion of complement regulatory protein genes, such as hCD46, hCD55, and hCD59, and genes to regulate the coagulation pathway or immune cell-mediated rejection may be required for an ideal xenotransplantation model. However, the technique for stable and efficient expression of multi-transgenes has not yet been settled to develop a suitable xenotransplantation model. To develop a stable and efficient transgenic system, we knocked-in internal ribosome entry sites (IRES)-mediated transgenes into the α 1,3-galactosyltransferase (GGTA1) locus so that expression of these transgenes would be controlled by the GGTA1 endogenous promoter. We constructed an IRES-based polycistronic hCD55/hCD39 knock-in vector to target exon4 of the GGTA1 gene. The hCD55/hCD39 knock-in vector and CRISPR/Cas9 to target exon4 of the GGTA1 gene were co-transfected into white yucatan miniature pig fibroblasts. After transfection, hCD39 expressed cells were sorted by FACS. Targeted colonies were verified using targeting PCR and FACS analysis, and used as donors for somatic cell nuclear transfer. Expression of GalT, hCD55, and hCD39 was analyzed by FACS and western blotting. Human complement-mediated cytotoxicity and human antibody binding assays were conducted on peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs), and deposition of C3 by incubation with human complement serum and platelet aggregation were analyzed in GGTA1 knock-out (GTKO)/CD55/CD39 pig cells. We obtained six targeted colonies with high efficiency of targeting (42.8% of efficiency). Selected colony and transgenic pigs showed abundant expression of targeted genes (hCD55 and hCD39). Knocked-in transgenes were expressed in various cell types under the control of the GGTA1 endogenous promoter in GTKO/CD55/CD39 pig and IRES was sufficient to express downstream expression of the transgene. Human IgG and IgM binding decreased in GTKO/CD55/CD39 pig and GTKO compared to wild-type pig PBMCs and RBCs. The human complement-mediated cytotoxicity of RBCs and PBMCs decreased in GTKO/CD55/CD39 pig compared to cells from GTKO pig. C3 was also deposited less in GTKO/CD55/CD39 pig cells than wild-type pig cells. The platelet aggregation was delayed by hCD39 expression in GTKO/CD55/CD39 pig. In the current study, knock-in into the GGTA1 locus and GGTA1 endogenous promoter-mediated expression of transgenes are an appropriable strategy for effective and stable expression of multi-transgenes. The IRES-based polycistronic transgene vector system also caused sufficient expression of both hCD55 and hCD39. Furthermore, co-transfection of CRISPR/Cas9 and the knock-in vector not only increased the knock-in efficiency but also induced null for GalT by CRISPR/Cas9-mediated double-stranded break of the target site. As shown in human complement-mediated lysis and human antibody binding to GTKO/CD55/CD39 transgenic pig cells, expression of hCD55 and hCD39 with ablation of GalT prevents an effective immunological reaction in vitro. As a consequence, our technique to produce multi-transgenic pigs could improve the development of a suitable xenotransplantation model, and the GTKO/CD55/CD39 pig developed could prolong the survival of pig-to-primate xenotransplant recipients.

LGR5 is a conserved marker of hair follicle stem cells in multiple species and is present early and throughout follicle morphogenesis

Hair follicle stem cells are key for driving growth and homeostasis of the hair follicle niche, have remarkable regenerative capacity throughout hair cycling, and display fate plasticity during cutaneous wound healing. Due to the need for a transgenic reporter, essentially all observations related to LGR5-expressing hair follicle stem cells have been generated using transgenic mice, which have significant differences in anatomy and physiology from the human. Using a transgenic pig model, a widely accepted model for human skin and human skin repair, we demonstrate that LGR5 is a marker of hair follicle stem cells across species in homeostasis and development. We also report the strong similarities and important differences in expression patterns, gene expression profiles, and developmental processes between species. This information is important for understanding the fundamental differences and similarities across species, and ultimately improving human hair follicle regeneration, cutaneous wound healing, and skin cancer treatment.

Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage.

As a member of the PIKs family, PIK3C3 participates in autophagy and plays a central role in liver function. Several studies demonstrated that the complete suppression of PIK3C3 in mammals can cause hepatomegaly and hepatosteatosis. However, the function of PIK3C3 overexpression on the liver and other organs is still unknown. In this study, we successfully generated PIK3C3 transgenic pigs through somatic cell nuclear transfer (SCNT) by designing a specific vector for the overexpression of PIK3C3. Plasmid identification was performed through enzyme digestion and transfected into the fetal fibroblasts derived from Diannan miniature pigs. After 2 weeks of culturing, six positive colonies obtained from a total of 14 cell colonies were identified through PCR. One positive cell line was selected as the donor cell line for SCNT for the construction of PIK3C3transgenic pigs. Thirty single blastocysts were collected and identified as PIK3C3 transgenic-positive blastocysts. Two surrogates became pregnant after transferring the reconstructed embryos into four surrogates. Fetal fibroblasts of PIK3C3-positive fetuses identified through PCR were used as donor cells for SCNT to generate PIK3C3 transgenic pigs. To further explore the function of PIK3C3 overexpression, genotyping and phenotyping of the fetuses and piglets obtained were performed by PCR, immunohistochemical, HE, and apoptosis staining. The results showed that inflammatory infiltration and vacuolar formation in hepatocytes and apoptotic cells, and the mRNA expression of NF-κB, TGF-β1, TLR4, TNF-α, and IL-6 significantly increased in the livers of PIK3C3 transgenic pigs when compared with wild-type (WT) pigs. Immunofluorescence staining showed that LC3B and LAMP-1-positive cells increased in the livers of PIK3C3 transgenic pigs. In the EBSS-induced autophagy of the porcine fibroblast cells (PFCs), the accumulated LC3II protein was cleared faster in PIK3C3 transgenic (PFCs) thanWT (PFCs). In conclusion, PIK3C3 overexpression promoted autophagy in the liver and associated molecular mechanisms related to the activation of ULK1, AMBR1, DRAM1, and MTOR, causing liver damage in pigs. Therefore, the construction of PIK3C3 transgenic pigs may provide a new experimental animal resource for liver diseases.

Validation of reliable safe harbor locus for efficient porcine transgenesis.

Transgenic technology is now widely used in biomedical and agricultural fields. Transgenesis is commonly achieved through random integration which might cause some uncertain consequences. The site-specific integration could avoid this disadvantage. This study aimed to screen and validate the best safe harbor (SH) locus for efficient porcine transgenesis. First, the cells carrying the EGFP reporter construct at four different SH loci (ROSA26, AAVS1, H11 and COL1A1) were achieved through CRSIPR/Cas9-mediated HDR. At the COL1A1 and ROSA26 loci, a higher mRNA and protein expression of EGFP was detected, and it was correlated with a lower level of DNA methylation of the EGFP promoter, hEF1α. A decreased H3K27me3 modification of the hEF1α promoter at the COL1A1 locus was also detected. For the safety of transgenesis at different SH locus, we found that transgenesis could relatively alter the expression of the adjacent endogenous genes, but the influence was limited. We also did not observe any off-target cleavage for the selected sgRNAs of the COL1A1 and ROSA26 loci. In conclusion, the COL1A1 and ROSA26 were confirmed to be the best two SH loci with the COL1A1 being more competitive for porcine transgenesis. This work would greatly facilitate porcine genome engineering and transgenic pig production.

Porcine β-defensin 2 confers enhanced resistance to swine flu infection in transgenic pigs and alleviates swine influenza virus-induced apoptosis possibly through interacting with host SLC25A4

Swine influenza virus (SIV) not only brings about great economic losses on the global pig industry, it also poses a significant threat to the public health for its interspecies transmission capacity. Porcine β-defensin 2 (PBD-2) is a host defense peptide and our previous study has shown that PBD-2 inhibits proliferation of enveloped pseudorabies virus both in vitro and in transgenic (TG) mice. The aim of this study is to investigate the possible anti-SIV ability of PBD-2 in a TG pig model created in our previous study. The in-contact challenge trial demonstrated that overexpression of PBD-2 in pigs could efficiently alleviate SIV-associated clinical signs. The SIV titers quantified by EID50 in lung tissues of infected TG pigs were significantly lower than that of wild-type littermates. In vitro, the cell viability assay revealed that PBD-2 mainly interfered with viral entry and post-infection stages. It was further confirmed that PBD-2 could enter porcine tracheal epithelial cells. The proteins interacting with PBD-2 inside host cells were identified with immunoprecipitation and the pathways involved were analyzed. Results showed that PBD-2 could interact with pro-apoptotic solute carrier family 25 member 4 (SLC25A4), also known as adenine nucleotide translocase 1, and thereby inhibited SIV-induced cell apoptosis. The molecular docking analysis suggested that PBD-2 interacted with porcine SLC25A4 mainly through strong hydrogen binding, with the predicted binding affinity being −13.23 kcal/mol. Altogether, these indicate that PBD-2 protects pigs against SIV infection, which may result from its role as a SLC25A4 blocker to alleviate cell apoptosis, providing a novel therapeutic and prophylactic strategy of using PBD-2 to combat SIV.

A Doomed Technology? On Gene Editing in Bavarian Livestock Agriculture, Policy Field Conflicts and Responsible Research and Innovation

The emergence of CRISPR-Cas9 has recently, for the first time, rendered the large-scale genetic modification of livestock animals such as cows, pigs, and chickens possible. Novel editing targets range from genes that curb disease vulnerabilities, increase muscle mass, or convey hornlessness, to the development of transgenic pigs for medical use. In this article, we discuss the efforts of a transdisciplinary research consortium in Bavaria, Germany, to test the technical and social feasibility of using CRISPR-Cas9-based gene editing as a novel technology in Bavarian small- to medium-scale livestock agriculture. The consortium comprised life scientists, local breeding associations, legal scholars, and social scientists from Science &amp;amp; Technology Studies (STS) and aimed to promote Responsible Research and Innovation (RRI) for gene editing technologies. Research focused on gene editing applications that improved animal health and all editing targets were co-developed with local breeding associations to meet the situated needs of small- to medium-scale livestock farmers in Bavaria. In this article, we discuss why the agricultural stakeholders in the project, that is, the representatives of local breeding associations, considered that, despite the project's success in generating positive research outcomes, it would be unlikely that results will be implemented in Bavarian livestock agriculture. We describe this situation in terms of a tension between agendas in the science and technology policy field and in the agricultural policy field in Bavaria that impacts local farmers' ability to adopt gene editing technologies. We further discuss what it might mean for RRI practices if public stakeholders are unlikely to benefit from the outcomes of RRI practices due to policy field conflicts or other contextual constraints and how STS scholars and other social scientists involved in RRI projects could adjust their practices to possibly redistribute benefits.

Intravitreal Administration of Stanniocalcin-1 Rescues Photoreceptor Degeneration with Reduced Oxidative Stress and Inflammation in a Porcine Model of Retinitis Pigmentosa

To investigate the effect of stanniocalcin-1 (STC-1), a secreted polypeptide exhibiting multiple functions in cell survival and death, on photoreceptor degeneration in a porcine model of retinitis pigmentosa (RP). P23H transgenic pigs (TG P23H) and wild-type hybrid littermates were obtained from the National Swine Resource and Research Center. Human recombinant STC-1 was injected intravitreally every 2 weeks from postnatal day 15 (P15) to P75. The contralateral eye was injected with balanced salt solution as a control. Electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed to evaluate retinal function and morphology in vivo at P90. Retinal tissue was collected for histologic analysis and molecular assays to evaluate the antioxidative and anti-inflammatory mechanisms by which STC-1 may rescue photoreceptor degeneration. Intravitreal injection of STC-1 improved retinal function in TG P23H pigs with increased photopic and flicker ERG a- and b-wave amplitudes. Greater integrity of the ellipsoid zone (EZ) band on SD-OCT and morphologic rescue with preservation of cone photoreceptors were observed in STC-1-treated TG P23H pigs. STC-1 altered gene expression in TG P23H pig retina on microarray analysis and increased photoreceptor specific gene expression by reverse transcription-polymerase chain reaction analysis. STC-1 significantly decreased oxidative stress and the expressions of NLRP3 inflammasome, cleaved caspase-1, and IL-1β in TG P23H pig retina. Intravitreal administration of STC-1 enhances cone photoreceptor function, improves EZ integrity, and reduces retinal degeneration through antioxidative and anti-inflammatory effects in a large animal (pig) model of the most common form of autosomal dominant RP in the United States.

Generation of reproductive transgenic pigs of a CRISPR-Cas9-based oncogene-inducible system by somatic cell nuclear transfer.

Alternative cancer models that are close to humans are required to create more valuable preclinical results during oncology studies. Here, a new onco-pig model via developing a CRISPR-Cas9-based Conditional Polycistronic gene expression Cassette (CRI-CPC) system to control the tumor inducing simian virus 40 large T antigen (SV40LT) and oncogenic HRASG12V . After conducting somatic cell nuclear transfer (SCNT), transgenic embryos were transplanted into surrogate mothers and five male piglets were born. Umbilical cord analysis confirmed that all piglets were transgenic. Two of them survived and they expressed a detectable green fluorescence. The test was made whether CRI-CPC models were naturally fertile and whether the CRI-CPC system was stably transferred to the offspring. By mating with a normal female pig, four offspring piglets were successfully produced. Among them, only three male piglets were transgenic. Finally, their applicability was tested as cancer models after transduction of Cas9 into fibroblasts from each CRI-CPC pig in vitro, resulting in cell acquisition of cancerous characteristics via the induction of oncogene expression. These results showed that our new CRISPR-Cas9-based onco-pig model was successfully developed.

OCT guided micro-focal ERG system with multiple stimulation wavelengths for characterization of ocular health

Inherited retinal disorders and dry age-related macular degeneration are characterized by the degeneration and death of different types of photoreceptors at different rate and locations. Advancement of new therapeutic interventions such as optogenetics gene therapy and cell replacement therapies are dependent on electrophysiological measurements at cellular resolution. Here, we report the development of an optical coherence tomography (OCT) guided micro-focal multi-color laser stimulation and electroretinogram (ERG) platform for highly localized monitoring of retina function. Functional evaluation of wild type and transgenic pigs affected by retinal degeneration was carried out using OCT guided micro-focal ERG (μfERG) with selected stimulation wavelengths for S, M and L cones as well as rod photoreceptors. In wild type pigs, μfERG allowed functional recording from rods and each type of cone photoreceptor cells separately. Furthermore, functional deficits in P23H transgenic pigs consistent with their retinal degeneration phenotype were observed, including decrease in the S and M cone function and lack of rod photoreceptor function. OCT guided μfERG based monitoring of physiological function will enable characterization of animal models of retinal degenerative diseases and evaluation of therapeutic interventions at the cellular level.

Transcriptomic analysis elucidates the enhanced skeletal muscle mass, reduced fat accumulation, and metabolically benign liver in human follistatin-344 transgenic pigs

Follistatin (FST) is an important regulator of skeletal muscle growth and adipose deposition through its ability to bind to several members of the transforming growth factor-β (TGF-β) superfamily, and thus may be a good candidate for future animal breeding programs. However, the molecular mechanisms underlying the phenotypic changes have yet to be clarified in pig. We generated transgenic (TG) pigs that express human FST specifically in skeletal muscle tissues and characterized the phenotypic changes compared with the same tissues in wild-type pigs. The TG pigs showed increased skeletal muscle growth, decreased adipose deposition, and improved metabolism status (P<0.05). Transcriptome analysis detected important roles of the PIK3–AKT signaling pathway, calcium-mediated signaling pathway, and amino acid metabolism pathway in FST-induced skeletal muscle hypertrophy, and depot-specific oxidative metabolism changes in psoas major muscle. Furthermore, the lipid metabolism-related process was changed in adipose tissue in the TG pigs. Gene set enrichment analysis revealed that genes related to lipid synthesis, lipid catabolism, and lipid storage were down-regulated (P<0.01) in the TG pigs for subcutaneous fat, whereas genes related to lipid catabolism were significantly up-regulated (P<0.05) in the TG pigs for retroperitoneal fat compared with their expression levels in wild-type pigs. In liver, genes related to the TGF-β signaling pathway were over-represented in the TG pigs, which is consistent with the inhibitory role of FST in regulating TGF-β signaling. Together, these results provide new insights into the molecular mechanisms underlying the phenotypic changes in pig.

Transgenic pigs expressing near infrared fluorescent protein-A novel tool for noninvasive imaging of islet xenotransplants.

Islet xenotransplantation is a promising concept for beta-cell replacement therapy. Reporter genes for noninvasive monitoring of islet engraftment, graft mass changes, long-term survival, and graft failure support the optimization of transplantation strategies. Near-infrared fluorescent protein (iRFP) is ideal for fluorescence imaging (FI) in tissue, but also for multispectral optoacoustic tomography (MSOT) with an even higher imaging depth. Therefore, we generated reporter pigs ubiquitously expressing iRFP. CAG-iRPF720 transgenic reporter pigs were generated by somatic cell nuclear transfer from FACS-selected stable transfected donor cells. Neonatal pig islets (NPIs) were transplanted into streptozotocin-diabetic immunodeficient NOD-scid IL2Rgnull (NSG) mice. FI and MSOT were performed to visualize different numbers of NPIs and to evaluate associations between signal intensity and glycemia. MSOT was also tested in a large animal model. CAG-iRFP transgenic NPIs were functionally equivalent with wild-type NPIs. Four weeks after transplantation under the kidney capsule, FI revealed a twofold higher signal for 4000-NPI compared to 1000-NPI grafts. Tenweeks after transplantation, the fluorescence intensity of the 4000-NPI graft was inversely correlated with glycemia. After intramuscular transplantation into diabetic NSG mice, MSOT revealed clear dose-dependent signals for grafts of 750, 1500, and 3000 NPIs. Dose-dependent MSOT signals were also revealed in a pig model, with stronger signals after subcutaneous (depth ∼6mm) than after submuscular (depth ∼15mm) placement of the NPIs. Islets from CAG-iRFP transgenic pigs are fully functional and accessible to long-term monitoring by state-of-the-art imaging modalities. The novel reporter pigs will support the development and preclinical testing of novel matrices and engraftment strategies for porcine xeno-islets.