Efficacy of the porcine species in biomedical research

1The first evidence of swine domestication dates back to approximately 7000–9000 years ago (;;;; Figure 1A). China and Europe have been, since domestication, the pig-breeding centers dictating the profile of the pig breeds. The reason for domestication was to provide meat as a source of food protein, which stimulated pig selection and farming. Studies have been conducted using genome-wide genotyping and genetic variability to trace the migration, selection, and improvement from ancient wild species to modern swine. It is generally accepted that the majority of all modern breeds are derived from the Eurasian wild boar (European and Asian wild boars;; ). Although pig selection started just after domestication, it has only been since the mid-20th century that performance has been used as the main tool in the animal selection process. More recently, molecular biology technologies, genome-wide association studies, and next-generation sequencing have been applied to enhance the selection process of domesticated pig breeds (e.g., Duroc, Landrace, Pietrain, Yorkshire, etc.) to further improve traits of high economic value such as feed conversion, meat quality, growth, precocious puberty, and prolificity.

2The variety of modern pig breeds available today, are a product of human intervention since domestication, but especially during the last century (Figure 1A). Besides breeds specialized for food production, smaller sized breeds (miniature- and micro-pigs) with certain characteristics such as obedience, friendly nature, and cognitive ability have also been selected for the purpose of companion animals. In addition, their use in biomedical research has been increasing considerably in the last years (Figure 1B).

3Compared with other animals used in research (e.g., mice, rats, rabbits, and dogs), domestic farm pigs are much larger (>300 kg adult size), therefore, requiring more space and feed, and making them harder to handle. Mini- or micro-pigs are hence more desirable for research use. The adult sizes vary among breeds, reaching around 20–30 kg for a Panepinto micropig to 100 kg for a Munich minipig. Although many minipig breeds are a product of crossbreeding, some breeds, like the Yucatan pigs, are naturally occurring stocks. Since the late 1940s, minipigs have been further developed specifically for biomedical research purposes.

4There are now several minipig breeds available for use in research. The main breeds developed in the USA are Yucatan, Sinclair (also known as Minnesota or Hormel miniature pig), Hanford, NIH minipig and Panepinto miniature pig. The minipig breeds developed in Europe are Göttingen, Munich, Berlin, Mini-Lewe, Czech-Republic, Vietnamese potbellied and Mini-Sib. In Asia, the breeds include Ohmini, Clawn, Lee Sung, and Chinese minipigs. The Göttingen and Yucatan breeds are the most commonly used minipigs in research, although there is no apparent clear reason for preference. Unlike the Yucatan, a natural breed, the Göttingen minipig was developed specifically for research use. Other breeds are used only by specific research groups, thus limiting their widespread availability in research. Nevertheless, the interest in the use of pigs in biomedical research has been rising over the last 40–45 years (Figure 1B).

5Biomedical research is broad, spanning studies on underlying disease mechanisms to the evaluation of safety and effectiveness of preventative measures, diagnostic tests, and therapies. Most animal studies in recent times have used the murine species due to their small size, fast reproductive cycles and short lifespan. In addition, the availability of murine embryonic stem cells, fully annotated genome, and facile tools for targeted genetic manipulation have all contributed to the elucidation of gene functions and disease pathophysiology. However, in many cases, mouse models do not adequately represent features of human disorders. In this regard, animals that better represent human pathophysiology are required. Pigs and humans share many similarities such as size, physiology, anatomy, metabolic profile, and longer lifespan. For example, pig skin is structurally similar to human skin regarding thickness and spacing between hair follicles, making it useful for studies on wound healing and burn lesions. Pigs also share anatomical and physiological similarities with respect to the renal system, making them valuable for pharmacological studies. Pigs can also be useful in the study of nutrient absorption and intestinal transport, as well as the pathogenesis of gastrointestinal diseases. All these characteristics contribute to the development of superior models of human conditions.

6The choice between outbred or inbred strains can have a significant impact on research outcomes. While, outbred strains may be better suited for quantitative trait loci studies, experiments addressing mechanistic aspects would benefit from the use of inbred strains. Some minipig breeds are already established for specific applications due to their unique characteristics (Table 1). Pigs have also been used for testing new therapies, devices, and efficacy and safety of new drugs prior to human trials. For instance, a novel endovascular chemotherapy filter, designed to reduce circulatory drug excess in vitro, was successfully tested in pigs. As well, a new method for pediatric liver transplantation was validated using pigs. Regarding pharmacokinetic and cytotoxic tests, pigs have been used for testing topical skin formulations, and are considered a better choice compared to dogs for the study of drugs that are metabolized by the aldehyde oxidase (AOX1), N-acetyltransferase (NAT1 or NAT2) or cytochrome (CYP2C9-like) enzymes.

7In general, there is low incidence of naturally occurring pathologies described in pigs. The reason for this is twofold. First, human intervention by way of selective breeding has eliminated genes that increased disease susceptibility. Second, the majority of the domestic farm pigs are slaughtered at a young age (< 6 months old), precluding the detection of late onset diseases such as cancer. On the other hand, Vietnamese potbellied minipigs raised as companion animals do reach old ages. Indeed, a retrospective study found a variety of neoplasms with widespread metastases in these pigs of advanced age (~11 years; ). The most common malignances found included hepatic and intestinal carcinomas, and uterine and ovarian smooth muscle tumors.

8Occurrence of malignant spontaneously regressing melanomas has been described in Sinclair minipigs. Selective interbreeding, by removing animals with red coat color that do not develop the lesions, increased the frequency of tumor formation in these selected minipigs. The tumors appear from birth and culminate in skin depigmentation after tumor regression showing a phenotype similar to human vitiligo. Studies conducted in these minipigs have shown decreased telomerase activity during melanoma regression, which has also been observed by inhibiting telomerase activity in human melanoma cells. Therefore, these minipigs may represent a useful model to study malignant melanomas because the tumors appear spontaneously and then either regress or grow progressively and metastasize similarly to human melanomas.

9Another example of a naturally occurring condition in pigs is the dwarf phenotype, caused by a single amino acid change in the α1 chain of type X collagen. The COL10A1 gene, which encodes type X collagen, is expressed in hypertrophic chondrocytes during endochondral ossification. In humans, an amino acid variation in the same position of the type X collagen protein has been shown to be the cause of Schmid metaphyseal chondrodysplasia (SMCD), a mild skeletal disorder associated with dwarfism. Since mice lacking type X collagen do not develop abnormalities in long bone development, pigs represent a better animal model of human SMCD.

10Another naturally occurring disease observed in Yucatan minipigs mimics human ventricular septal defect (VSD; ). The VSD in pigs can be observed in fetal stages similar to the congenital anomaly in humans, and can be used for the study of new methods of diagnosis or therapies.

11Despite a number of natural occurring pig phenotypes that resemble human diseases, for most of human pathologies it is difficult to find representative animal models in nature. Thus, manipulation of diet, use of drugs and/or surgeries has been necessary to generate appropriate models. For example, minipig models for Type I diabetes were induced via administration of streptozotocin or alloxan to selectively destroy insulin-producing cells. High-energy diets in young minipigs lead to the development of obesity and metabolic syndromes, with increased visceral fat deposition, glucose intolerance, decreased insulin sensitivity, and higher levels of blood cholesterol and triglycerides, which progress to Type 2 Diabetes mellitus. Other chemicals have been used to induce cellular dysregulation and damage in pigs including the administration of N-nitrosodiethylamine to produce a liver cancer model.

12Genetically modified animals have been instrumental in advancing our understanding of gene function and significance of inappropriate gene expression in metabolic malfunction in mammals. Genome editing holds great promise in generating these models, and has already permitted the rapid development of new pig models of several human diseases.

13The cystic fibrosis (CF) model is an example of genetically engineered pigs created by targeted inactivation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The resulting pigs exhibit clinical features and disease progression consistent with those observed in CF infants. In contrast, inactivation of the CFTR gene in mice did not produce the comorbidities typically observed in human CF patients.

14Advanced reproductive technologies, such as somatic cell nuclear transfer (SCNT), can now be routinely applied to large animal species, including minipigs. Minipigs of different breeds have been cloned from different cell types, including genetically modified cells. In addition SCNT offers the possibility of creating isogenic and immunocompatible animals from the same cell line. Importantly, models of severe disorders can be generated from engineered cultured cells without the need of breeding sick animals. The sequencing of the pig genome is another key development in the production of gene-modified pigs in the post-genomic era. Genome editing techniques, including zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered, regularly interspaced, short palindromic repeats (CRISPR) together with CRISPR associated (Cas) nucleases (CRISPR/Cas), now allow the precise manipulation of gene sequences in germ, embryonic and somatic cells. Among these methods, the CRISPR/Cas9 system is emerging as the method of choice because it permits gene editing to be accomplished in only one step by injecting both the specific guide RNAs and endonuclease into zygotes.

15Another example of human disease that has the potential to be studied in genetically engineered pigs is heart arrhythmias. Mutations in the SCN5A gene, which encodes a subunit of the cardiac sodium channel Nav1.5 required for excitability and conduction in the myocardium, were found in patients with Bruguda syndrome. SCN5AE558X/+ engineered Yucatan minipigs with reduced expression of the sodium channel Nav1.5 have been created and these animals exhibit conduction abnormalities and susceptibility to ventricular arrhythmias. There has also been considerable interest in genetically modified pig strains suitable for xenotransplantation. Most research into the development of appropriate xenotransplantation strains focused on addressing hyperacute rejection, which is initiated rapidly and involves preformed natural human antibodies and the complement system. This has been possible by targeting cell surface antigens such as α-1,3 galactosyltransferase or complement regulatory proteins such as human decay accelerating factor. The pigs made deficient of α-1,3 galactosyltransferase have contributed to the reduction of immunogenicity of donor tissue/organs. Transgenic pigs expressing antibodies against cytotoxic T-cell lymphocyte antigen receptor, a cell-mediated immune response suppressor, were also developed.

16A pig model for the human familial adenomatous polyposis was generated by inactivation of the adenomatous polyposis coli (APC) gene. Mice lacking the APC gene exhibit non-metastatic neoplasias only in the small intestine. However, the pig model of colon and rectal cancer reproduces the human features of the disease, which includes the development of polyps spread along the whole large bowel in young animals. A candidate gene for the development of breast and ovarian cancer models is the breast cancer-associated gene 1 (BRCA1), which has been manipulated in both Yucatan and Göttingen cells, but lines of modified minipigs remain to be produced. The TP53 gene, which encodes the tumor suppressor protein p53 and is the most commonly observed suppressed gene in human tumors, was found to be mutated in Li-Fraumeni patients having increased risk to develop multiple types of cancers. Suppression of p53 in mesenchymal stem cells derived from pig bone marrow exhibits chemoresistance in vitro. Mutation of TP53 gene in Yucatan minipigs resulted in development of lymphomas and osteogenic tumors. More recently, a new engineered pig strain termed “oncopig” was developed, which promises inducible formation of a wide variety of cancers that are potentially novel platforms for research and therapeutics development. These examples illustrate the potential of genetically engineered pigs as robust models for the study of human pathologies that are not well represented in small laboratory animal species.

17Rodents have been the choice animal model for basic research, but are not always suitable for translational research due to marked differences in size, lifespan as well as metabolic, anatomical, and physiological discrepancies. On the other hand, the pig is more closely related to humans in terms of these parameters and, therefore, is better suited for recapitulation of human diseases. Indeed, the use of the pig in translational research is increasingly gaining acceptance (Figure 1C). Dogs and non-human primates have traditionally been used for this purpose, but rising ethical concerns have reduced their favor and increased demand for alternatives. The number of peer-reviewed papers describing the use of pigs as biomedical models has risen eightfold over the past 30 years (Figure 1B). Already, the pig has become well established in many areas of research and training. For instance, in the past 20 years the pig has replaced the dog as a model for surgical training and has also gained FDA approval for the testing of surgical implantation devices intended for human use. Minipig models, which are much smaller in size compared to the domestic farm breeds, offer lower operating costs compared to other large animal models and also reduce the concern of ethical acceptance given the already widespread use of pigs in agriculture.

18Pigs offer many exciting applications, including stem cell research, tissue engineering and xenotransplantation. Although incredible advances in transgenic pigs harboring various engineered alterations designed to minimize graft versus host rejection, much work remains to be accomplished since multiple genes need to be manipulated given the various types of tissue rejection reactions. Porcine induced pluripotent stem cells (iPSCs) have been produced and chimeric pigs were generated using iPSC. This is highly relevant since study of porcine iPSCs have eventual human applications, such as cell-based therapies. However, the mechanisms of cellular reprogramming, directed cell differentiation and species-specific cell culture requirements necessitate further investigation. The International Society for Stem Cell Research has indicated in their guidelines for translational use that validation must occur in both small and large animal models. Tissue repair is another potential application of engineered pig models. Cartilage tissue grafts have been created using chondrocytes isolated from infant minipigs, and mandibular condyle grafts have been generated from Yucatan minipig adipose-derived mesenchymal stem cells. There has also been successful regeneration of bone defects using engineered bone graft tissues in minipig models. If custom donor transgenic minipig strains can be created, this could open the doors to other engineered tissue replacements for human uses. For example, the use of blastocyst complementation and pluripotent stem cells has been applied to direct the development of otherwise missing organs in pigs. This has increased the hope that it may one day be possible to create non-immunogenic donor organs in pigs using human iPSCs. Finally, similarities in the porcine and human immune system have sparked interest in vaccine development and efficacy testing in pigs.

19The completion of the porcine genome project in 2012 has further facilitated the use of pigs in research. Data from this project has enabled the comparative analysis of genetic sequences and development of the necessary tools to create and validate targeted genetic alterations in the porcine genome. In addition, the development of RNASeq technology has facilitated transcriptome analysis, which further improves our ability to identify important targets related to certain phenotypic traits. Other recent achievements in the pig include the use of inducible or conditional systems to control transgene expression, and tissue-specific expression of the Cre recombinase. These advances will ensure the continued development of various pig strains for research, similar to what has already been accomplished in mice.

20It is clear that the use of the pig as a biomedical model is increasingly gaining approval due to physiopathological similarities with humans. However, some obstacles remain to be overcome in order to realize the full potential of the porcine species in developing new diagnostic and therapeutic approaches. Despite the sequencing of the porcine genome, full annotation has yet to be completed. This is essential to facilitate interrogation of the pig genome and investigation of less characterized genes. Efforts to develop a complete porcine proteome map as well as epigenome map are currently underway. These databases are necessary to understand disease pathogenesis. Moreover, the availability of both inbred and outbred breeds of minipigs extends the utility of these species as a viable large animal model. Continuing refinements and adaptation of technologies for genome editing, cell/tissue-specific gene targeting strategies, stem cells and somatic cell cloning will further facilitate the creation of specialized pig strains for biomedical research.

21The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.