Neonatal islets from human PD-L1 transgenic pigs reduce immune cell activation and cellular rejection in humanized nonobese diabetic-scid IL2rγnull mice.

Strong xenorejection limits the clinical application of porcine islet transplantation in type 1 diabetes. Targeting T cell-mediated rejection is one of the main approaches to improve long-term graft survival. Here we study engraftment and survival of porcine islet cells expressing human programmed cell death ligand-1 (hPD-L1) in a humanized mouse model. Neonatal islet-like clusters (NPICCs) from transgenic hPD-L1 (hPD-L1-Tg) and wild-type (Wt) pigs were transplanted into nonobese diabetic-scid IL2rγnull mice stably reconstituted with human immune cells (hPD-L1 n = 10; Wt n = 6). Primary endpoint was development of normoglycemia during a 16-week observation period after transplantation. Secondary endpoints were porcine C-peptide levels and immune cell infiltration. Animals transplanted with hPD-L1-Tg neonatal islet-like clusters achieved a superior normoglycemic rate (50% versus 0%) and significantly higher plasma C-peptide levels as compared to the Wt group, indicating long-term beta cell function. Intracytoplasmic fluorescence-activated cell sorting analysis and immunohistochemistry revealed significantly decreased frequencies of interferonγ-expressing splenic hCD8-positive T cells and reduced intragraft-infiltrating immune cells. We here demonstrate that expression of hPD-L1 provides strong islet xenograft protection without administration of immunosuppressive drugs. These findings support the hypothesis that hPD-L1 has the capacity to control cellular rejection and therefore represents a very promising transgene candidate for clinical porcine islet xenotransplantation.

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.

Targeted overexpression of PPARγ in skeletal muscle by random insertion and CRISPR/Cas9 transgenic pig cloning enhances oxidative fiber formation and intramuscular fat deposition.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis and lipogenesis. To understand its roles in fiber formation and fat deposition in skeletal muscle, we successfully generated muscle-specific overexpression of PPARγ in two pig models by random insertion and CRISPR/Cas9 transgenic cloning procedures. The content of intramuscular fat was significantly increased in PPARγ pigs while had no changes on lean meat ratio. PPARγ could promote adipocyte differentiation by activating adipocyte differentiating regulators such as FABP4 and CCAAT/enhancer-binding protein (C/EBP), along with enhanced expression of LPL, FABP4, and PLIN1 to proceed fat deposition. Proteomics analyses demonstrated that oxidative metabolism of fatty acids and respiratory chain were activated in PPARγ pigs, thus, gathered more Ca2+ in PPARγ pigs. Raising of Ca2+ could result in increased phosphorylation of CAMKII and p38 MAPK in PPARγ pigs, which can stimulate MEF2 and PGC1α to affect fiber type and oxidative capacity. These results support that skeletal muscle-specific overexpression of PPARγ can promote oxidative fiber formation and intramuscular fat deposition in pigs.

Delayed body development with reduced triglycerides levels in leptin transgenic pigs.

Leptin is a well-known adipokine that plays critical role in adiposity. To further investigate the role of leptin in adiposity, we utilized leptin overexpressing transgenic pigs and evaluated the effect of leptin on growth and development, fat deposition, and lipid metabolism at tissue and cell level. Leptin transgenic pigs were produced and divided into two groups: elevated leptin expression (leptin ( +)) and normal leptin expression group (control). Results indicated that leptin ( +) pigs had elevated leptin protein and mRNA expression levels and exhibited sluggish growth and development followed by decreased subcutaneous fat thickness, low serum triglycerides, saturated, unsaturated fatty acids and high cholesterol esters (p < 0.05). There were differences in the lipid metabolism related genes at different fat depots, including upregulation of PPARγ, AGPAT6, PLIN2, HSL and ATGL in subcutaneous, PPARγ in perirenal, and FAT/CD36 and PLIN2 in mesenteric adipose tissues and downregulation of AGPAT6 and ATGL in perirenal and AGPAT6 in mesenteric adipose tissues (p < 0.05). Additionally, in-vitro cultured leptin ( +) preadipocytes exhibited upregulation of PPARγ, FAT/CD36, ACACA, AGPAT, PLIN2, ATGL and HSL as compared to control (p < 0.05). These findings suggested that homeostasis imbalance in lipolysis and lipogenesis at adipose tissue and adipocytes levels led to low subcutaneous fat depots in leptin overexpression pigs. These pigs can act as model for obesity and related metabolic disorder.

hEPCR.hTBM.hCD47.hHO-1 with donor clodronate and DDAVP treatment improves perfusion and function of GalTKO.hCD46 porcine livers perfused with human blood.

INTRODUCTION: Platelet sequestration, inflammation, and inappropriate coagulation cascade activation are prominent in liver xenotransplant models and are associated with poor outcomes. Here, we evaluate a cassette of six additional genetic modifications to reduce anti-pig antibody binding (α-1,3-galactosyl transferase knockout [GalTKO]) and target coagulation dysregulation (human endothelial protein C receptor [hEPRC] and thrombomodulin [hTBM]), complement pathway regulation (human membrane cofactor protein, hCD46), inflammation heme oxygenase 1 [hHO-1]), and a self-recognition receptor (integrin-associated protein [hCD47]), as well as donor pharmacologic treatments designed to blunt these phenomena. METHODS: Livers from GaltKO.hCD46 pigs ("2-gene," n = 3) and GalTKO.hCD46 pigs also transgenic for hEPRC, hTBM, hCD47, and hHO-1 ("6-gene," n = 4) were perfused ex vivo with whole human blood. Six-gene pigs were additionally pretreated with desmopressin (DDAVP) and clodronate liposomes to deplete vWF and kupffer cells, respectively. RESULTS: The average perfusion times increased from 304 (±148) min in the 2-gene group to 856 (±61) min in the 6-gene group (p = .010). The average heparin administration was decreased from 8837 U/h in the 2-gene to 1354 U/h in the 6-gene group (p = .047). Platelet sequestration tended to be delayed in the 6-gene group (p = .070), while thromboxane B2 (TXB2, a platelet activation marker) levels were lower over the first hour (p = .044) (401 ± 124 vs. 2048 ± 712 at 60 min). Thrombin production as measured by F1+2 levels tended to be lower in the 6-gene group (p = .058). CONCLUSIONS: The combination of the hEPCR.hTBM.hCD47.hHO-1 cassette along with donor pig DDAVP and clodronate liposome pretreatment was associated with prolonged function of xenoperfused livers, reduced coagulation pathway perturbations, and decreased TXB2 elaboration, and reflects significant progress to modulate liver xenograft injury in a pig to human model.

Regulatory changes in China on xenotransplantation and related products.

BACKGROUND: Given the persistence and the worldwide shortage of organs from both the deceased and living donors for clinical transplantation, pig organs or tissues hold immense promises for the patients on the waiting list, and xenotransplantation is deemed as one of the solutions to the organ shortage crisis. Indeed, the emerging gene editing technologies, such as CRISPR/Cas9, have led to tremendous progress in the generation of genetically modified pigs to overcome many barriers associated. METHOD: We presented a description of the xenotransplantation regulations in China and the related products. RESULT: Several groups in China have successfully generated transgenic pigs with the elimination of immune rejection or coagulation-related genes, and both pre-clinical and clinical studies have been reported. However, the pre-clinical evaluation and clinical application of porcine xenotransplantation raises ethical and regulatory considerations. Herein, in this review, we will summarize and discuss the progress in xenotransplantation in China and xenotransplantation-related products from the regulatory perspective. CONCLUSION: There has been remarkable progress in both the genetically modified pigs and pre-clinical studies in China, and China will be the first country to successfully fulfill the xenotransplantation from pig organ to human in the near future.

ß-Glucanase specific expression in the intestine of transgenic pigs.

Producing heterologous enzymes in the animal digestive tract to improve feed utilization rate is a new research strategy by transgenic technology. In this study, transgenic pigs specifically expressing ß-glucanase gene in the intestine were successfully produced by somatic cell nuclear transfer technology in order to improve digestibility of dietary ß-glucan and absorption of nutrients. The ß-glucanase activity in the intestinal juice of 4 transgenic pigs was found to be 8.59 ± 2.49 U/mL. The feeding trial results showed that the crude protein digestion of 4 transgenic pigs was significantly increased compared with that of the non-transgenic pigs. In order to investigate the inheritance of the transgene, 7 G1 transgenic pigs were successfully obtained. The ß-glucanase activity in the intestinal juice of 7 G1 transgenic pigs was found to be 2.35 ± 0.72 U/mL. The feeding trial results showed the crude protein digestion and crude fat digestion were significantly higher in 7 G1 transgenic pigs than in non-transgenic pigs. Taken together, our study demonstrated that the foreign ß-glucanase expressing in the intestine of the transgenic pigs could reduce the anti-nutritional effect of ß-glucans in feed. In addition, ß-glucanase gene could be inherited to the offsprings and maintain its physiological function. It is a promising approach to improve feed utilization by producing transgenic animals.

Co-expression of fat1 and fat2 in transgenic pigs promotes synthesis of polyunsaturated fatty acids.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are essential for the development and health of mammals, such as humans and livestock. n-3 PUFAs must be supplied by diet due to the absence of a key gene, namely, delta-15 desaturase (fat1), which is responsible for synthesizing n-3 PUFAs from a major type of n-6 PUFAs, linoleic acid (LA). To increase the dietary intake of n-3 PUFAs for humans, fat1-expressing transgenic (TG) livestock have been produced to provide n-3 PUFA-rich meats for humans. However, these TG livestock synthesized n-3 PUFAs from diet-derived, instead of endogenously produced, n-6 PUFAs because they still lack the delta-12 desaturase (fat2) gene for catalyzing conversion of internal oleic acid (OA) to LA. To fill the gap in the de novo n-3 PUFA biosynthesis pathway and to increase n-3 PUFA content in livestock, TG pigs co-expressing fat1-fat2 were generated in the present work. The OA content decreased in fat1-fat2 TG pigs, suggesting that OA was converted to LA by fat2 transgene-encoded delta-12 desaturase. The n-3 PUFA level was elevated, and the n-6/n-3 PUFA ratio dropped in fat1-fat2 TG pigs, revealing that fat1 transgene promoted the synthesis of n-3 PUFAs from n-6 analogs. The expression levels of fatty acid elongase-5 (ELOVL5) and fatty acid elongase-2 (ELOVL2), which are two key enzyme genes for PUFA synthesis, as well as their transcription factor peroxisome proliferator-activated receptor α, increased in fat1-fat2 TG pigs. Thus, the fat1 transgene enhanced n-3 PUFA synthesis by upregulating the expression of enzyme genes involved in the PUFA synthesis pathways. Overall, this study provided a new strategy to produce n-3 PUFA-rich meat for human consumption. The generated fat1-fat2 TG pigs can also serve as a large animal model for studying the roles of n-3 PUFAs in human development and health.

Transgenic pigs expressing ß-xylanase in the parotid gland improve nutrient utilization.

Xylan is one of the main anti-nutritional factors in pig's feed. Although supplementation of ß-xylanase in diet can improve the utilization of nutrients in animals, it is limited by feed cost, manufacturing process and storage stability. To determine whether the expression of endogenous ß-xylanase gene xynB in vivo can improve digestibility of dietary xylan and absorption of nutrients, we produced transgenic pigs which express the xynB from Aspergillus Niger CGMCC1067 in the parotid gland via nuclear transfer. In four live transgenic founders, ß-xylanase activities in the saliva were 0.74, 0.59, 0.37 and 0.24 U/mL, respectively. Compared with non-transgenic pigs, the content of crude protein (CP) in feces reduced by 15.5% (P < 0.05). Furthermore, in 100 of the 271 F1 pigs the xynB gene was detectable. The digestibility of gross energy and CP in F1 transgenic pigs were increased by 5% and 22%, respectively, with the CP content in feces decreased by 6.4%. Taken together, our study showed that the transgenic pigs producing ß-xylanase from parotid gland can reduce the anti-nutritional effect in animal diet and improve the utilization of nutrients.

Production of genetically modified pigs expressing human insulin and C-peptide as a source of islets for xenotransplantation.

Islet xenotransplantation is a promising treatment for type I diabetes. Numerous studies of islet xenotransplantation have used pig-to-nonhuman primate transplantation models. Some studies reported long-term survival and successful function of porcine islets in diabetic monkeys. Genetic engineering techniques may improve the survival and function of porcine islets. A recent study reported the generation of transgenic pigs expressing human insulin rather than porcine insulin by changing one amino acid at the end of the ß-chain in insulin. However, C-peptide from pigs still existed. In this study, we generated transgenic pigs expressing human proinsulin to express human insulin and C-peptide using fibroblasts from proinsulin knockout pigs as donor cells for somatic cell nuclear transfer. Eleven live piglets were delivered from three surrogates and characterized to confirm the genotype and phenotype of the generated piglets. Genotype analysis of the generated piglets showed that five of the eleven piglets contained the human proinsulin gene. Insulin expression was confirmed in the serum and pancreas in two of the five piglets. C-peptide derived from human proinsulin was also confirmed by liquid chromatography tandem mass spectrometry. Non-fasting blood glucose level was measured to verify the function of the insulin derived from the human proinsulin. Two piglets expressing insulin showed normal glucose levels similar to that in the wild-type control. In conclusion, human insulin- and C-peptide-expressing pigs without porcine insulin and C-peptide were successfully established. These pigs can be used as a source of islets for islet xenotransplantation.

Generation of insulin-deficient piglets by disrupting INS gene using CRISPR/Cas9 system.

Diabetes mellitus is a chronic disease with accompanying severe complications. Various animal models, mostly rodents due to availability of genetically modified lines, have been used to investigate the pathophysiology of diabetes. Using pigs for diabetic research can be beneficial because of their similarity in size, pathogenesis pathway, physiology, and metabolism with human. However, the use of pigs for diabetes research has been hampered due to only few pig models presenting diabetes symptoms. In this study, we have successfully generated insulin-deficient pigs by generating the indels of the porcine INS gene in somatic cells using CRISPR/Cas9 system followed by somatic cell nuclear transfer. First, somatic cells carrying a modified INS gene were generated using CRISPR/Cas9 system and their genotypes were confirmed by T7E1 assay; targeting efficiency was 40.4% (21/52). After embryo transfer, three live and five stillborn piglets were born. As expected, INS knockout piglets presented high blood glucose levels and glucose was detected in the urine. The level of insulin and c-peptide in the blood serum of INS knockout piglets were constant after feeding and the expression of insulin in the pancreas was absent in those piglets. This study demonstrates effectiveness of CRISPR/Cas9 system in generating novel pig models. We expect that these insulin-deficient pigs can be used in diabetes research to test the efficacy and safety of new drugs and the recipient of islet transplantation to investigate optimal transplantation strategies.

Pigs expressing the human inhibitory ligand PD-L1 (CD 274) provide a new source of xenogeneic cells and tissues with low immunogenic properties.

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.

Synthetic liver function is detectable in transgenic porcine livers perfused with human blood.

In addition to immune barriers, molecular incompatibilities between species are predicted to limit pig liver survival in primate xenotransplantation models. Assessment and measurement of synthetic function of genetically modified porcine livers after ex vivo perfusion with human blood have not previously been described. Eight porcine livers from α1,3-galactosyl transferase knockout and human membrane cofactor (GalTKO.hCD46), six livers from GalTKO.hCD46 and N-glycolylneuraminic acid knockout (GalTKO.hCD46.Neu5GcKO), and six livers from GalTKO.hCD46 with humanized decay-accelerating factor (hCD55), endothelial protein C receptor (hEPCR), tissue factor pathway inhibitor (hTFPI), and integrin-associated protein (hCD47) (GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47) pigs were perfused with human blood under physiologic conditions. Timed blood samples were tested for liver enzymes and for pig-specific albumin production via Western blot. Porcine albumin levels increased with time in all experiments. By densitometry, GalTKO.hCD46.Neu5GcKO livers had the highest albumin levels, measured both as total produced, and when controlled for perfusion duration, compared to GalTKO.hCD46 (P = .068) and GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47 livers (P = .04). Porcine livers perfused with human blood demonstrated the synthetic ability to produce albumin in all cases. GalTKO.hCD46.Neu5GcKO pig livers demonstrated the most robust albumin production. This suggests that the Neu5GcKO phenotype provides a protective effect on the graft due to decreased human antibody recognition and graft injury.

Release of pig leukocytes and reduced human NK cell recruitment during ex vivo perfusion of HLA-E/human CD46 double-transgenic pig limbs with human blood.

BACKGROUND: In pig-to-human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non-classical human leukocyte antigen-E (HLA-E) on pEC. Thus, the aim of this study was to analyze NK cell responses to vascularized xenografts using an ex vivo perfusion system of pig limbs with human blood. METHODS: Six pig forelimbs per group, respectively, stemming from either wild-type (wt) or HLA-E/hCD46 double-transgenic (tg) animals, were perfused ex vivo with heparinized human blood for 12 hours. Blood samples were collected at defined time intervals, cell numbers counted, and peripheral blood mononuclear cells analyzed for phenotype by flow cytometry. Muscle biopsies were analyzed for NK cell infiltration. In vitro NK cytotoxicity assays were performed using pEC derived from wt and tg animals as target cells. RESULTS: Ex vivo, a strong reduction in circulating human CD45 leukocytes was observed after 60 minutes of xenoperfusion in both wt and tg limb groups. NK cell numbers dropped significantly. Within the first 10 minutes, the decrease in NK cells was more significant in the wt limb perfusions as compared to tg limbs. Immunohistology of biopsies taken after 12 hours showed less NK cell tissue infiltration in the tg limbs. In vitro, NK cytotoxicity against hCD46 single tg pEC and wt pEC was similar, while lysis of double tg HLA-E/hCD46 pEC was significantly reduced. Finally, circulating cells of pig origin were observed during the ex vivo xenoperfusions. These cells expressed phenotypes mainly of monocytes, B and T lymphocytes, NK cells, as well as some activated endothelial cells. CONCLUSIONS: Ex vivo perfusion of pig forelimbs using whole human blood represents a powerful tool to study humoral and early cell-mediated rejection mechanisms of vascularized pig-to-human xenotransplantation, although there are several limitations of the model. Here, we show that (i) transgenic expression of HLA-E/hCD46 in pig limbs provides partial protection from human NK cell-mediated xeno responses and (ii) the emergence of a pig cell population during xenoperfusions with implications for the immunogenicity of xenografts.

The aflatoxin-detoxifizyme specific expression in the parotid gland of transgenic pigs.

Producing aflatoxin-detoxifizyme (ADTZ) in pigs to control the AFT contamination of pig feed is a new research strategy by transgenic technology. In this study, transgenic pigs specifically expressing ADTZ gene in the parotid gland were successfully produced by somatic cell nuclear transfer technology. The ADTZ activity in saliva of 6 transgenic pigs was found to be 7.11 ± 2.63 U/mL. The feeding trial with aflatoxin (AFT) results showed that there were significant difference about the serum biochemical index such as total protein (TP), albumin (ALB), globulin (GLB) contents and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity and AFT residues in serum and liver between the pigs in the test treatment (transgenic pigs) producing ADTZ and those in the positive control (P < 0.05). In order to investigate the inheritance of the transgene, 11 G1 transgenic pigs were successfully obtained. The ADTZ activity in saliva of 11 G1 transgenic pigs was found to be 5.82 ± 1.53 U/mL. The feeding trial with AFT results showed that the serum biochemical index containing TP, ALB and GLB contents and ALT and AST activity and AFB1 residues in serum and liver of the pigs in the test treatment (transgenic pigs) producing ADTZ were significantly different than those in the positive control (P < 0.05). The above results demonstrated that ADTZ produced in transgenic pigs could improve the effect of the AFT contamination of feed on pigs.

Generation of α-1,3-galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes.

Recent progress in genetic manipulation of pigs designated for xenotransplantation ha6s shown considerable promise on xenograft survival in primates. However, genetic modification of multiple genes in donor pigs by knock-out and knock-in technologies, aiming to enhance immunological tolerance against transplanted organs in the recipients, has not been evaluated for health issues of donor pigs. We produced transgenic Massachusetts General Hospital piglets by knocking-out the α-1,3-galactosyltransferase (GT) gene and by simultaneously knocking-in an expression cassette containing five different human genes including, DAF, CD39, TFPI, C1 inhibitor (C1-INH), and TNFAIP3 (A20) [GT-(DAF/CD39/TFPI/C1-INH/TNFAIP3)/+] that are connected by 2A peptide cleavage sequences to release individual proteins from a single translational product. All five individual protein products were successfully produced as determined by western blotting of umbilical cords from the newborn transgenic pigs. Although gross observation and histological examination revealed no significant pathological abnormality in transgenic piglets, hematological examination found that the transgenic piglets had abnormally low numbers of platelets and WBCs, including neutrophils, eosinophils, basophils, and lymphocytes. However, transgenic piglets had similar numbers of RBC and values of parameters related to RBC compared to the control littermate piglets. These data suggest that transgenic expression of those human genes in pigs impaired hematopoiesis except for erythropoiesis. In conclusion, our data suggest that transgenic expression of up to five different genes can be efficiently achieved and provide the basis for determining optimal dosages of transgene expression and combinations of the transgenes to warrant production of transgenic donor pigs without health issues.

Improvement of anti-nutritional effect resulting from ß-glucanase specific expression in the parotid gland of transgenic pigs.

ß-Glucan is the predominant anti-nutritional factors in monogastric animal feed. Although ß-glucanase supplementation in diet can help to eliminate the adverse effects, enzyme stability is substantially modified during the feed manufacturing process. To determine whether the expression of endogenous ß-glucanase gene (GLU) in vivo can improve digestibility of dietary ß-glucan and absorption of nutrients, we successfully produced transgenic pigs via nuclear transfer which express the GLU from Paenibacillus polymyxa CP7 in the parotid gland. In three live transgenic founders, ß-glucanase activities in the saliva were 3.2, 0.07 and 0.03 U/mL, respectively, and interestingly the enzyme activities increased in the pigs from 178 days old to 789 days old. From the feed the amount of gross energy, crude protein and crude fat absorbed by the transgenic pigs was significantly higher than the non-transgenic pigs. Meanwhile the moisture content of the feces was significantly reduced in transgenic pigs compared with the non-transgenic pigs. Furthermore, in all positive G1 pigs, ß-glucanase activity was detectable and the highest enzyme activity reached 3.5 U/mL in saliva. Also, crude protein digestion was significantly higher in G1 transgenic pigs than in control pigs. Taken together, our data showed that the transgenic ß-glucanase exerted its biological catalytic function in vivo in the saliva, and the improved performance of the transgenic pigs could be accurately passed on to the offspring, indicating a promising alternative approach to improving nutrient availability was established to improve utilization of livestock feed through transgenic animals.

Direct introduction of gene constructs into the pronucleus-like structure of cloned embryos: a new strategy for the generation of genetically modified pigs.

Due to a rising demand of porcine models with complex genetic modifications for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a novel strategy for the generation of genetically modified pigs, termed "nuclear injection". To evaluate the reliability of this new strategy, the developmental ability of embryos in vitro and in vivo as well as the integration and expression efficiency of a transgene carrying green fluorescence protein (GFP) were examined. Eighty percent of the cloned pig embryos (633/787) exhibited a PN-like structure, which met the prerequisite to technically perform the new method. GFP fluorescence was observed in about half of the total blastocysts (21/40, 52.5%), which was comparable to classical zygote PN injection (28/41, 68.3%). In total, 478 cloned embryos injected with the GFP construct were transferred into 4 recipients and from one recipient 4 fetuses (day 68) were collected. In one of the fetuses which showed normal development, the integration of the transgene was confirmed by PCR in different tissues and organs from all three primary germ layers and placenta. The integration pattern of the transgene was mosaic (48 out of 84 single-cell colonies established from a kidney were positive for GFP DNA by PCR). Direct GFP fluorescence was observed macro- and microscopically in the fetus. Our novel strategy could be useful particularly for the generation of pigs with complex genetic modifications.

Characterization of Growth and Reproduction Performance, Transgene Integration, Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer.

Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance and characterized the transgene insertion, transmission, and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression, and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favorable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition.

Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-to-primate kidney transplant model.

Xenotransplantation has the potential to alleviate the organ shortage that prevents many patients with end-stage renal disease from enjoying the benefits of kidney transplantation. Despite significant advances in other models, pig-to-primate kidney xenotransplantation has met limited success. Preformed anti-pig antibodies are an important component of the xenogeneic immune response. To address this, we screened a cohort of 34 rhesus macaques for anti-pig antibody levels. We then selected animals with both low and high titers of anti-pig antibodies to proceed with kidney transplant from galactose-α1,3-galactose knockout/CD55 transgenic pig donors. All animals received T-cell depletion followed by maintenance therapy with costimulation blockade (either anti-CD154 mAb or belatacept), mycophenolate mofetil, and steroid. The animal with the high titer of anti-pig antibody rejected the kidney xenograft within the first week. Low-titer animals treated with anti-CD154 antibody, but not belatacept exhibited prolonged kidney xenograft survival (>133 and >126 vs. 14 and 21 days, respectively). Long-term surviving animals treated with the anti-CD154-based regimen continue to have normal kidney function and preserved renal architecture without evidence of rejection on biopsies sampled at day 100. This description of the longest reported survival of pig-to-non-human primate kidney xenotransplantation, now >125 days, provides promise for further study and potential clinical translation.