Editorial

Engineering bone phenotypes in domestic animals: Unique resources for enhancing musculoskeletal research

The genetic engineering of domestic animals has increased significantly in recent years, particularly with the advent of CRISPR-Cas9 gene editing tchnology. The utility of genome editing technology provides a unique opportunity for musculoskeletal investigators to consider the examination of rare phenotypes in domestic animals or perhaps more so, to develop domestic animal models of the bone disorders in which the bone remodeling process more closely resembles that observed in humans. However, the generation of large animal models requires specialized animal husbandry facilities and demands improved analytical capabilities. Accurately assessing the molecular and cellular bone phenotype of a particular genotype is not a simple task and requires the characterization of large bone biopsy specimens, the development of ex vivo culture systems from different species as well as the availability of non-invasive imaging for larger animals. Once characterized, a specific phenotype will contribute to various purposes resulting in new mechanistic insights bone remodeling, specific details of muscle-bone-tendon interactions and perhaps also provide an opportunity for improved treatment and/or therapeutic interventions that are mechanism-based. In this perspective, we interrogate the modeling of bone phenotypes in domestic animals and evaluate the role of these species in the growth of the musculoskeletal field, considering the high current preference for rodent models. Whatever the eventual outcome, it is clear that recent biotechnology developments in gene editing and the annotation of more genomes will undoubtedly bring significant changes to the way musculoskeletal phenotypes are developed, studied and evaluated.

Historically, genetically modified mice, in particular mice in which a specific gene has been inactivated (knockouts), have served as a cornerstone for models of animal and human disease and as the ultimate test for the determination of gene function. However, mice are not completely representative of human physiology, metabolism, genetics, lifespan, or size and many times engineered mice do not exhibit the same phenotype or reveal the same gene function(s) observed in humans. Large animal models of disease often offer distinct advantages because they are in many ways more representative of human physiology and represent alternative solutions to issues with genetic testing of gene action. Genetic engineering of livestock is not a new concept but has become increasingly more efficient and thus more cost effective of late, making the utilization of these valuable large animal research resources available to scientists across multiple disciplines (Table 1). It is important to note that many other livestock species disease models exist but limited space prevented the inclusion of an exhaustive list

The first genetically modified livestock were produced in the 1980s, with the primary focus being agricultural applications, reviewed by Pursel [1]. These initial experiments involved genetic engineering of sheep and pigs to improve production traits such as feed efficiency and meat quality [[2], [3], [4]]. Results of these experiments verified the usefulness of genetically engineered livestock and laid the groundwork for future research. These important studies ultimately resulted in the production of a large number of different genetically engineered animals, such as pigs resistant to various viral diseases, pigs that produce phytase to facilitate the efficient digestion of phosphorous to decrease pollution (Enviropig ™), hornless cattle, cattle with increased muscle development, goats that produce milk with a longer shelf life, as well as chickens resistant to avian influenza [[5], [6], [7], [8], [9], [10], [11]].

While the animals listed above were produced primarily to benefit production agriculture, i.e. to be used as food, other genetically engineered livestock were developed with the goal of producing therapeutics, pharmaceuticals and supplements that could be used to treat human and/or animal disease. ATryn® (antithrombin) was the first approved recombinant therapeutic which is an anticoagulant produced in the milk of transgenic goats [12]. More recently, Pharming's Ruconest®, a C1-estrase inhibitor used for the treatment of hereditary angioedema, provided a second complete case study for the development of drugs from transgenic rabbits [13]. Many other livestock species (cows, sheep, goats, pigs and rabbits) have been engineered to produce therapeutic proteins in milk. These include alpha antitrypsin to treat cystic fibrosis, lactoferrin, used to treat stomach and intestinal ulcers, diarrhea and hepatitis C, albumin to treat burn patients, and recombinant human butyrylcholinesterase to treat oganophosphate poisoning, just to name a few. Transgenic cows which produce human antibodies in their blood have also been produced and promise to provide therapeutic approaches to treat a wide variety of human diseases [7,[14], [15], [16], [17], [18], [19], [20]].

Livestock production and utilization as models for biomedical research have become increasing available due to improved technical efficiency. The broad utility of livestock transgenics was dramatically altered after the nuclei of somatic cells from an adult mammal were used to create “Dolly” [21] and shortly thereafter Polly [22]. Genetic engineering has proven extremely useful in enabling animals to produce novel therapeutic proteins [23], and this rapidly evolved to include models of human disease. These pioneering days of genetic engineering, driven almost entirely by insertion of large gene constructs into the animal genome (transgenics), have been superseded with recent advances in the field [24,25]. The new technologies do not solely involve transgenesis and in fact allows for the generation of targeted approaches to genetically engineer animals via gene deletion or by the specific manipulation of sequences within endogenous genes. Given the large and almost daily expansion of sequenced genomes, there is now unprecedented access to detailed sequence information, including control regions, coding regions, and known allelic variants in all the major livestock species as well as the specific gene editing technology needed to modify gene function [26].

The new technologies of gene editing have been added to the molecular toolbox for genetic manipulation of various organisms. Gene editing involves the utilization of a number of DNA modifying enzymes such as zinc-finger proteins (ZFP) [27], transcription activator-like effector nuclease (TALENS) [28] or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) [29]. All of these operate in basically the same manner by binding to specific genomic locations and inducing DNA strand breaks. ZFPs and TALENS rely on engineered protein domains to recognize DNA, which is then cut by fused FokI nucleases whereas the CRISPR system uses Watson-Crick base pairing with single-stranded RNA to recognize a target sequence that is then cut by a Cas nuclease. Once this occurs, natural DNA repair mechanisms take over and in many cases the repair event results in non-homologous end joining (NHEJ) which causes a mutation at the specific cut site. When a mutation occurs, the target gene is modified and in the case of inactivation, unable to function. Alternatively, the system can be used in conjunction with the addition of a new sequence designed for targeted insertion into the cut site via homology directed repair (HDR). Gene editing is applicable to many different organisms including mice and livestock and has proven to be remarkably efficient [[30], [31], [32], [33], [34], [35], [36]].

Of all the gene editing platforms, application of the CRISPR-Cas9 system has become the method of choice for establishing animal models of disease [35,37]. Indeed, CRISPR-Cas9 is now widely accepted as a simple and versatile RNA-directed system for genome editing in a wide range of different organisms and cell types, including bacteria, mice, rat, zebrafish, human cells, and a variety of livestock species [35,37]. Yet to date, the genetically engineered phenotypes generated in domestic animals have almost entirely been for improved production traits [[38], [39], [40]], novel/therapeutic protein expression [23], or for models of human disease [[41], [42], [43]] but with little specific focus on the musculoskeletal system.

These advances in genome engineering along with a desire to utilize improved models of human disease has led to significant interest in developing gene edited large animal models. Given these broad genetic capabilities are now widely available, the almost singular focus of the bone field on murine models of bone disease is in urgent need of revision. With the increasing availability of reliable genomic sequence information for domestic animals (goats, sheep, pigs and cattle) and the ubiquitous gene manipulation tools, increasing numbers of genetically engineered livestock models, such as sheep, are appearing and being utilized in biomedical research [44,45] (Table 1). This perspective discusses the potential use of genetically engineered domestic animals in bone biology to highlight the beneficial characteristics of large animal models of human disease that complement the widespread utility of available rodent models.