Genetically Modified Pigs Research Articles

Genome editing pig models with elements for controllable gene expression

AbstractConditional gene regulation systems can control gene expression in predefined tissues or organs at a desired time. Site‐specific recombinase systems and chemically induced gene expression systems are the two most widely used approaches for creating genetically modified (GM) animals with conditional regulation of gene expression. Generation of GM pigs with controllable elements, usually involving multiple gene editing, used to be a major challenge due to a lack of germ line‐competent pluripotent stem cells. With the emergence of artificial endonuclease‐mediated gene editors, a variety of GM pigs with recombinase‐specific recognition elements or chemically induced elements for conditional regulation of gene expression have been generated by the combination of site‐directed knock‐in of somatic cells and somatic cell nuclear transfer technology, allowing conditional deletion of endogenous genes or overexpression of exogenous genes in pigs. These inducible tool pig models will greatly facilitate the production of GM pigs and broaden the applications of transgenic pigs in biomedicine and agriculture fields. In this paper, we review the progress in the construction and application of pigs with controllable elements using gene editing techniques.

Co-expression of HLA-E and HLA-G in Porcine Endothelial Cells Attenuates Human Natural Killer Cell-Mediated Cytotoxicity

INTRODUCTION: Pig-to-human xenotransplantation (XTx) is a promising solution to the organ shortage. Genetically-modified (GM) pigs lacking major xenoantigens have reduced hyperacute rejection and prolonged xenograft survival. Despite these advancements, acute rejection remains a barrier to clinical XTx. Natural killer cells (NK) play a crucial role in transplantation. In the context of XTx, suppressing human NK activity by expressing HLA class I molecules in pig organs may protect xenografts from immune attack. Therefore, we aimed to co-express HLA class I molecules in GM pig endothelial cell line null for 5 key xenoantigens (5GKO) and to evaluate human NK response to 5GKO+HLA class I+ cells. METHODS: DNA sequences encoding HLA-E and HLA-G were designed, synthesized, and cloned into expression vectors. Three GM porcine cell lines (5GKO.HLA-E, 5GKO.HLA-G, 5GKO.HLA-E+G) were generated by transfection. IL-2–activated human peripheral blood mononuclear cells (PBMCs) from 5 donors were co-cultured with GM porcine cell lines to initiate NK cell–mediated immune responses. NK activation (CD107a expression) was evaluated by flow cytometry. Three experimental replicates were averaged and analyzed by one-way ANOVA and a post hoc Tukey test. RESULTS: We successfully co-expressed HLA-G, HLA-E, and HLA-E+G in 5GKO cells. Co-expression of HLA-E+G in 5GKO cells significantly reduced human NK activation compared with 5GKO, 5GKO.HLA-E, and 5GKO.HLA-G. CONCLUSION: Co-expression of HLA-E and HLA-G in porcine cells significantly reduces NK cell–mediated cytotoxicity and shows promise for incorporation into GM pigs for pig-to-human xenotransplantation.

The Renin‐Angiotensin System after Pig Kidney Transplantation in Baboons

Resolution of many technical and immune‐related issues have allowed long‐term survival of baboons with life‐supporting pig kidney grafts. However, episodes of hypovolemia and hypotension during the post‐transplant period occur via an unexplained mechanism. It has been reported that pig renin has low reactivity on human angiotensinogen (AGT). Because the renin‐angiotensin system (RAS) is vitally important in the regulation of extracellular fluid volume and blood pressure, we evaluated the activity of the RAS in baboon’s post‐pig kidney xenotransplantation. Kidneys from genetically‐modified pigs were transplanted into 5 baboons (6 to 10 kg) that were immunosuppressed by blockade of the CD40/CD154 costimulation pathway, after excision of the native kidneys. Plasma renin, AGT (only baboon AGT could be measured), and angiotensin II levels were measured by ELISAs in (i) healthy genetically‐modified pigs (n=3), healthy non‐immunosuppressed baboons (n=6), and (iii) immunosuppressed baboons with pig kidney grafts. Samples were collected for up to 120 days post‐transplantation. Plasma renin levels were significantly greater in healthy pigs (586±139 (SE) pg/ml) than in intact baboons (430±28 pg/ml). The plasma renin levels in baboons with pig kidney grafts were similar to values in intact baboons (367±18 pg/ml), indicating the continuing production of renin by the pig kidney grafts in absence of baboon kidneys. Plasma AGT (which reflected only baboon AGT) levels were augmented in the transplanted baboons (21±0.8 µg/ml) compared to the pre‐transplant concentrations (9.9±0.8). Plasma angiotensin II levels were 16±1.3 pg/ml in intact baboons and trended lower from pre‐transplant levels in the transplanted baboons (10±1.6 pg/ml). Lower plasma angiotensin II concentrations in baboons with pig kidney grafts, even in the presence of increasing plasma AGT levels, may be a consequence of reduced, but not absent, reactivity of pig renin to cleave baboon AGT. The reduced angiotensin II levels in the transplanted baboons suggest an impaired ability to elicit a robust response to hypovolemic and hypotensive episodes.

Consumer responses to genetically modified food in China: The influence of existing general attitudes, affect and perceptions of risks and benefits

Identifying the factors that influence people’s acceptance of genetically modified (GM) food can inform industries and policymakers regarding their innovation trajectories, and policy development and implementation. The relative importance of existing general attitudes towards GM food, and of affective responses to specific GM food applications in shaping associated risk perceptions and acceptability, has been infrequently assessed in empirical research. The present study used an online survey with Chinese consumers (n = 1,411) to explore how general attitudes and affect interact and inform people’s responses to three GM food applications. The results of structural equation modelling indicated the perceived benefits of GM foods were the most important predictor of acceptability. General attitudes and affect both had larger impacts on acceptability compared to risk perceptions, with general attitudes having a larger effect for the GM yeast and GM soybean and affect exerting a stronger influence for the GM pig. General attitudes also had a moderating effect on the relationship between risk perceptions and application acceptability, reinforcing the systemic cognitive bias that generalises general attitudes-induced a priori judgements to all three GM food applications. When respondents associated GM applications with tampering with nature, perceived unnaturalness and risk perceptions both increased. Collectively, the results indicate that the design of communication about GM food applications should incorporate the influence of affect invoked by the reported benefits and risks of applications, alongside accommodating the potential influence of the recipients’ existing general attitudes.

Scientific and psychosocial ethical considerations for initial clinical trials of kidney xenotransplantation.

The initial clinical trials of pig solid organ xenotransplantation (XTx) are drawing closer and could begin in the coming years. The first clinical trials may aim to transplant genetically-modified pig kidneys into adult humans. The impetus for beginning these first-in-human trials is the severe lack of deceased donor kidneys for transplantation and the number of patients with end-stage renal disease currently on transplant waitlists, which in the USA approaches 100000. The majority of patients on the kidney transplant waitlist receive continuous renal replacement therapy. In the United States, for patients on the kidney waitlist, the median wait-time to receive a deceased human donor organ is approximately 4.5 years for patients aged 45-74, with a 5-year mortality (or removal from the waitlist because of deteriorating health) of approximately 40%. XTx has the potential to reduce the kidney waitlist morbidity and mortality while improving quality of life. By focusing on scientific and psychosocial criteria, we present ethical considerations of certain inclusion and exclusion criteria for these first-in-human clinical trials that we suggest have not yet been fully explored.

Impact of donor and prolonged cold ischemia time of neonatal pig pancreas on neonatal pig islet transplant outcome.

Genetically modified pigs (GMP) have been developed to alleviate the shortage of donors in human islet transplantation and rejection. In this study, we characterized and compared the islets from GalTKO, GalTKO/hCD46, GalTKO/hCD46/hCD39, and wild-type (WT) neonatal pigs. Islets were isolated from GMP and WT pig pancreases that have been packaged with ice pack for at least 24hours. The difference in gene expression and function of islets were evaluated by microarray analysis and transplantation of islets under the kidney capsule of streptozotocin-induced diabetic immune-deficient mice, respectively. Blood glucose levels of these mice were monitored weekly post-transplantation for >100days, and islet grafts were collected and evaluated for the presence of endocrine cells. The genes involved in extracellular components, cell adhesion, glucose metabolism, and inflammatory response are differentially expressed between GMP and WT pig islets. Variation in the ability of pig islets in correcting the diabetic state of the mouse recipients appears to be dependent on the pig donor. In addition, prolonged cold ischemia time had a negative effect on the transplant outcome. All normoglycemic mice were able to respond well to glucose challenge despite the initial differences in the ability of islet transplants to reverse their diabetic state. Islet xenografts of normoglycemic mice contained abundant insulin- and glucagon-positive cells. The effect of GMP and WT neonatal pig islet transplants on hyperglycemia in mice appears to be dependent on the pig donor, and prolonged cold ischemia time negatively affects the neonatal pig islet transplant outcome.

Efficient deletion of LoxP-flanked selectable marker genes from the genome of transgenic pigs by an engineered Cre recombinase.

Genetically modified (GM) pigs hold great promises for pig genetic improvement, human health and life science. When GM pigs are produced, selectable marker genes (SMGs) are usually introduced into their genomes for host cell or animal recognition. However, the SMGs that remain in GM pigs might have multiple side effects. To avoid the possible side effects caused by the SMGs, they should be removed from the genome of GM pigs before their commercialization. The Cre recombinase is commonly used to delete the LoxP sites-flanked SMGs from the genome of GM animals. Although SMG-free GM pigs have been generated by Cre-mediated recombination, more efficient and cost-effective approaches are essential for the commercialization of SMG-free GM pigs. In this article we describe the production of a recombinant Cre protein containing a cell-penetrating and a nuclear localization signal peptide in one construct. This engineered Cre enzyme can efficiently excise the LoxP-flanked SMGs in cultured fibroblasts isolated from a transgenic pig, which then can be used as nuclear donor cells to generate live SMG-free GM pigs harboring a desired transgene by somatic cell nuclear transfer. This study describes an efficient and far-less costly method for production of SMG-free GM pigs.

Establishment of a Decaplex PCR-Capillary Gel Electrophoresis Method for the Simultaneous Detection of Six Kinds of Genetically Modified Animals.

This study aimed to establish an event-specific multiplex PCR system using microsatellite markers and fluorescently labeled primers to detect six different genetically modified (GM) animal lines, including human lactoferrin GM cattle, human lysozyme GM cattle, human α-lactalbumin GM cattle, myostatin knockout pigs, phytase GM pigs, and ω-3 fatty acid desaturase gene GM pigs. Four different microsatellite loci for species identification, along with six GM animal-specific fragments, were selected as targets for primer design. The capillary gel electrophoresis results of multiplex PCR showed that the target fragments were amplified successfully. This high-throughput multiplex PCR detection system can be applied for the inspection and quarantine of GM animals.

Effect of ferulic acid supplementation on the developmental competence of porcine embryos during in vitro maturation.

The value of laboratory and genetically-modified pigs is becoming increasingly clear; however, their in vitro development remains inefficient. Trans-ferulic acid (trans-FA) is an aromatic compound that is abundant in plant cell walls, and which exhibits antioxidant effects in vitro. Trans-FA is known to improve sperm viability and motility; however, its effects on porcine oocytes are unknown. Our aim was to investigate the effects of trans-FA supplementation during in vitro maturation on the meiotic and developmental competence of porcine oocytes. Oocytes were matured either without (control) or with trans-FA (10, 100 and 1,000 µM), fertilized, and cultured in vitro for 7 days. The maturation rate of oocytes cultured with 10 µM trans-FA (81.6%) was significantly higher than that of controls (65.0%; P<0.05). The fertilization rate of oocytes matured with 10 µM trans-FA (57.4%) was also significantly higher than that of controls (32.7%) and oocytes cultured with other concentrations (33.1% and 22.7% for 100 and 1,000 µM, respectively; P<0.05). Moreover, the blastocyst formation rate of oocytes matured with 10 µM trans-FA (6.9%) was significantly higher than that of controls (2.3%; P<0.05). Our results suggest that in vitro maturation with 10 µM trans-FA is beneficial for the in vitro production of porcine embryos and has the potential to improve production system.

Genome editing revolutionize the creation of genetically modified pigs for modeling human diseases.

Pigs have anatomical, physiological and genomic characteristics that make them highly suitable for modeling human diseases. Genetically modified (GM) pig models of human diseases are critical for studying pathogenesis, treatment, and prevention. The emergence of nuclease-mediated genome editing technology has been successfully employed for engineering of the pig genome, which has revolutionize the creation of GM pig models with highly complex pathophysiologies and comorbidities. In this review, we summarize the progress of recently developed genome editing technologies, including zinc finger nucleases(ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), which enable highly efficient and precise introduction of genome modifications into pigs, and tailored disease models that have been generated in various disciplines via genome editing technology. We also summarize the GM pig models that have been generated by conventional transgenic strategies. Additionally, perspectives regarding the application of GM pigs in biomedical research are discussed.

Evaluation of human Cd69+ T-cell phenotypes after co-culture with genetically-modified pig mesenchymal stromal cells: An in vitro xenotransplantation model

Evaluation of human Cd69+ T-cell phenotypes after co-culture with genetically-modified pig mesenchymal stromal cells: An in vitro xenotransplantation model

Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System.

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP+/eGFP+) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

Human T cells upregulate CD69 after coculture with xenogeneic genetically-modified pig mesenchymal stromal cells

Mesenchymal stromal cells (MSC) obtained from α1,3-galactosyltransferase gene knock-out pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46 pMSC) suppress in vitro human anti-pig cellular responses as efficiently as allogeneic human MSC. We investigated the immunoregulatory effects of GTKO/CD46 pMSC on human CD4+ and CD8+ T cell proliferation in response to pig aortic endothelial cells (pAEC). pMSC efficiently suppressed T cell proliferation, which was associated with downregulation of granzyme B expression. No induction of CD4+CD25+Foxp3hi regulatory T cells or T cell apoptosis was documented. In correlation with T cell proliferation, CD25 expression was upregulated on T cells in response to pAEC but not to pMSC. In contrast, CD69 expression was upregulated on T cells in response to both pMSC and pAEC, which was associated with a significant increase in the phosphorylation of STAT5. GTKO/CD46 pMSC possibly regulate human T cell responses through modulation of CD69 expression and STAT5 signaling.

324 PRODUCTION OF GENETICALLY MODIFIED PIGS BY ARTIFICIAL REPRODUCTIVE TECHNOLOGIES USING FROZEN EPIDIDYMAL SPERM

Genetically modified (GM) pigs are useful tools for many types of biomedical research. The objective of this study was to develop a reliable protocol for the reproduction of GM pigs using frozen epididymal sperm. Epididymal sperm were collected from 3 GM boars: (1) humanized Kusabira-Orange (huKO) transgenic, (2) mutant human hepatocyte nuclear factor 1α (HNF-1α) transgenic, and (3) α1,3-galactosyltransferase gene knockout (GalT-KO). Additionally, a wild type (WT) boar was also used for sperm collection. After collection, the sperm were frozen in Niwa and Sasaki freezing medium (Niwa et al. 1989 Manual for Cryopreservation of Pig Spermatozoa 19–23; 1.0 × 109 sperm mL–1) using 0.5-mL straws. The sperm were cooled utilising the 2-step method, from 25 to 15°C at a rate of 0.17°C min–1, and then from 15 to 5°C at 0.08°C min–1. After cooling to 5°C, the straws were frozen in liquid nitrogen with 3% glycerol for storage. After storage for 0.5 to 21 months, the straws were thawed by immersion into a 37°C water bath. The recovered sperm were washed by centrifugation in PBS + 1 mg mL–1 BSA and were resuspended in either Beltsville thawing solution (Pursel and Johnson. 1975 J. Anim. Sci. 40, 99–102) for intra-fallopian AI or in porcine fertilization medium (PFM, Research Institute for the Functional Peptides, Yamagata, Japan) for IVF. Prepubertal gilts (6.5 months), in which oestrus was induced by 1000 IU eCG and 1500 IU hCG given 66 or 72 h apart, were surgically inseminated with 0.9–7.9 × 106 progressive motile sperm 41 to 51 h after receiving an hCG injection. Oocytes, which matured in vitro either in modified porcine oocyte medium (IFP) or modified NCSU23 medium, were inseminated in PFM for 8 h with either 0.5 (huKO, GalT-KO) or 1.6 (HNF-1α) × 106 progressive motile sperm mL–1. The in vitro fertilized oocytes were cultured in porcine zygote medium-5 (Research Institute for the Functional Peptides) for 5 to 6 days until they were transferred. Some of these oocytes were examined for their fertilization rates. Blastocysts were transferred into the uterus of the recipient 150 h after hCG treatment. The fertilization rates for huKO, HNF-1α and GalT-KO sperm were 63.5% (61/96), 62.5% (70/112) and 92.4% (61/66), respectively; 50.8% (31/61), 82.9% (58/70) and 60.7% (37/61) of these were monospermic. The rate at which the IVF embryos developed into blastocysts was 47.8–52.4%. This study demonstrates that IVF using frozen epididymal sperm is more efficient method for reproducing genetically modified pigs than AI (87.5 v. 33.3%). Table 1.Pig production efficiencies of frozen epididymal sperm from genetically modified pigs utilising AI and IVF This study was supported by JST, ERATO, NAKAUCHI Stem Cell and Organ Regeneration Project, Tokyo.

The Effective Multicistronic Expression System by a Single Vector Cassette Using the Viral 2A Segment

Introduction: The multiple protein expression using a single vector cassette is a very useful technique for generation of geneticallymodified pigs for xenotransplantation. The internal ribosomal entry site (IRES) element has a pitfall that the second gene at downstream is not expressed well relative to the first gene in mammalian cells. Although the second gene is well expressed in mammalian cells by the viral 2A vector system, the different destination of target proteins may affect the mutual expression level. Here, we investigated whether the upstream and downstream gene of the viral 2A segment is well expressed regardless of their final destination. Materials and methods: We cloned various cassettes including two genes into mammalian expression vector, pcDNA3.1 and transfected each of them into pig embryonic fibroblasts by electroporation. The protein expression levels were checked by western blotting or flow cytometry using extra- or intra-staining protocol. Results: In cassette # 1 and 2, the protein of EGFP and HA-HO1 at upstream of IRES element was expressed well. But, the protein expression of HA-HO1 and EGFP at downstream of IRES element was very low or not detected. In cassette # 3, 4, 5 and 6 the protein of EGFP, HA-HO1, EPCR and TM was expressed well regardless of their location in the vector. In cassette # 7 and 8, the protein of TM was expressed well regardless of the location. But, the protein expression of EGFP at the downstream of T2A segment was lower than the upstream EGFP. In cassette # 9, the protein expression level of the upstream gene, CD46, was extremely low and the the downstream gene, TM, was not expressed. In cassette # 10, the both of genes were expressed well. Overall, these data demonstrated that the multicistronic expression system using the viral 2A segment works better than the system using IRES element in mammalian cells, and that the protein expression level of target genes in the viral 2A system, was almost same regardless of their location in the vector even if they go toward different destination in cells. However, the protein expression level of the first gene determines the expression level of the second gene.Table: [Vector cassettes using in this study]Conclusion: The multicistronic expression system using the viral 2A segment is a useful system for making good expression of multiple genes, if protein expression level of the first gene is adequate. Therefore, a 2A vector system is promising for making genetically-modified pigs for multiple target genes for xenotransplantation. (Abbreviations: HA-HO1: Heme oxygenase 1 tagged with HA; TM: Thrombomodulin; EPCR: Endothelial protein C receptor; T2A: 2A segment from Thosea asigna virus; CP: Cytosolic protein; MP: Membrane protein)

Attitudes towards the use of genetically modified animals in research

Here we provide the first experimental evidence that public concerns about the use of animals in research are accentuated when genetically modified (GM) animals are used. Using an online survey, we probed participant views on two uses of pigs as research animals (to reduce agricultural pollution or to improve organ transplant success in humans) with and without GM. We surveyed 327 animal technicians, researchers, advocates, university students and others. In both scenarios and across demographics, support dropped off when the research required the use of GM pigs or GM corn. For example, 66% of participants supported using pigs to reduce phosphorus pollution, but this declined to 49% when the pigs were fed GM corn and to 20% when the research required the creation of a new GM line of pigs. Those involved in animal research were more consistently supportive compared to those who were not or those who were vegetarians.

The potential of genetically-modified pig mesenchymal stromal cells in xenotransplantation

Ezzelarab M, Ayares D, Cooper DKC. The potential of genetically-modified pig mesenchymal stromal cells in xenotransplantation.Xenotransplantation 2010; 17: 3–5. © 2010 John Wiley & Sons A/S.

Porcine cells express more than one functional ligand for the human lymphocyte activating receptor NKG2D

Xenotransplantation could ameliorate the severe shortage of donor organs. The initial results of transplantation from genetically-modified pig donors to primate recipients suggest that hyperacute rejection can be overcome, but thrombotic microangiopathy and the human anti-pig cellular immune response remain as significant impediments to successful clinical xenotransplantation. NKG2D is an activating immunoreceptor found on human natural killer (HuNK) cells, CD8(+) and gammadelta T cells. Signaling through NKG2D mediates cytotoxicity and cytokine secretion by NK cells and co-stimulation of T cells. Chinese hamster ovary P (CHOP) cells were transfected with human NKG2D and used in cell-cell binding studies with porcine epithelial, and endothelial cell lines. Soluble recombinant NKG2D-Fc was used to stain various porcine cells and tissues to indicate ligand expression. Porcine cells were used as targets in cytotoxicity assays with the HuNK cell lines NKL and YT, with and without enzymatic removal of pULBP1 and antibody blockade of NKG2D signaling. In this study, we demonstrate the expression of ligands for human NKG2D on porcine cell lines of endothelial and epithelial origin, islet cell clusters and rejecting kidney. HuNK cells were activated to kill pig cells expressing NKG2D ligands, and cytotoxicity was inhibited by antibody blockade of NKG2D. A previous study identified pULBP1 as the principal ligand for human NKG2D on pig aortic endothelial cells. In the current study, renal epithelial and intestinal endothelial cells each expressed high surface levels of pULBP1, but binding of soluble recombinant NKG2D and NKG2D-dependent cytotoxicity against these cells persisted after the enzymatic removal of pULBP1, strongly suggesting the presence of at least one additional functional ligand for human NKG2D in these cell types.

Use of molecular modeling and site-directed mutagenesis to define the structural basis for the immune response to carbohydrate xenoantigens.

BackgroundNatural antibodies directed at carbohydrates reject porcine xenografts. They are initially expressed in germline configuration and are encoded by a small number of structurally-related germline progenitors. The transplantation of genetically-modified pig organs prevents hyperacute rejection, but delayed graft rejection still occurs, partly due to humoral responses. IgVH genes encoding induced xenoantibodies are predominantly, not exclusively, derived from germline progenitors in the VH3 family. We have previously identified the immunoglobulin heavy chain genes encoding VH3 xenoantibodies in patients and primates. In this manuscript, we complete the structural analysis of induced xenoantibodies by identifying the IgVH genes encoding the small proportion of VH4 xenoantibodies and the germline progenitors encoding xenoantibody light chains. This information has been used to define the xenoantibody/carbohydrate binding site using computer-simulated modeling.ResultsThe VH4-59 gene encodes antibodies in the VH4 family that are induced in human patients mounting active xenoantibody responses. The light chain of xenoantibodies is encoded by DPK5 and HSIGKV134. The structural information obtained by sequencing analysis was used to create computer-simulated models. Key contact sites for xenoantibody/carbohydrate interaction for VH3 family xenoantibodies include amino acids in sites 31, 33, 50, 57, 58 and the CDR3 region of the IgVH gene. Site-directed mutagenesis indicates that mutations in predicted contact sites alter binding to carbohydrate xenoantigens. Computer-simulated modeling suggests that the CDR3 region directly influences binding.ConclusionXenoantibodies induced during early and delayed xenograft responses are predominantly encoded by genes in the VH3 family, with a small proportion encoded by VH4 germline progenitors. This restricted group can be identified by the unique canonical structure of the light chain, heavy chain and CDR3. Computer-simulated models depict this structure with accuracy, as confirmed by site-directed mutagenesis. Computer-simulated drug design using computer-simulated models may now be applied to develop new drugs that may enhance the survival of xenografted organs.