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. Author manuscript; available in PMC: 2018 Sep 1.
Published in final edited form as: Mol Reprod Dev. 2017 Jun 8;84(9):1012–1017. doi: 10.1002/mrd.22812

Genome-Editing Technologies to Improve Research, Reproduction, and Production in Pigs

Kevin D Wells 1, Randall S Prather 1,*
PMCID: PMC5603364  NIHMSID: NIHMS869609  PMID: 28394093

Abstract

The ability to directly manipulate the pig genome through genetic engineering has been available to the research community for over three decades. This technology has progressed from the random insertion of foreign DNA, via a variety of techniques (pronuclear microinjection, sperm mediated gene transfer, and integration of mobile genetic elements), to manipulation of endogenous genes, via homologous recombination in somatic cells followed by somatic cell nuclear transfer. Over the last few years, designer nucleases facilitated the development of techniques that provide efficient ways to introduce foreign DNA or to modify endogenous genes in eggs, zygotes, or somatic cells. Together, these genome-editing technologies have essentially removed the obstacles to gene manipulation in swine. Although the regulatory environment is still unclear for agricultural applications, genetic engineering of pigs will continue to advance biomedicine and biology. In addition, genetic engineering is now sufficiently simple and efficient that agricultural research can now ask basic and applied questions that are not hampered by limited funding.

Keywords: homologous recombination, translational research, CRISPR/Cas9, non-homologous end joining

Introduction

With very few exceptions, all cells within an animal have the same set of genes. The function of each cell is determined by the repertoire of genes transcribed within that cell. Genetic engineering provides the opportunity to alter these genes, thus affecting the transcriptome of a cell.

In the past, creating genomic changes required cumbersome and/or inefficient methods, and the imaginable genetic changes, while theoretically possible, proved impractical to create. The recent development of new tools has vastly improved the efficiency of making the desired, predetermined genetic changes. For example, the newest genetic engineering tools make the production of knockouts, domain swaps, and single-base changes an expected and highly efficient process.

The current array of tools available to alter gene expression is extensive, allowing us to modify the expression of individual genes in most any manner imaginable. Furthermore, as the general knowledge of biological phenomena expands, specific knowledge from one species can be co-opted as tools to study other species. Indeed, specific, sometimes esoteric, biological molecules can be reassembled and applied to practically any system of interest through molecular biology. In the end, biotechnology provides opportunities to elucidate intricacies that have thus far been indecipherable.

Techniques for Genetic Engineering

The swine genome is ~2.7 Gb packaged within 18 autosomes plus X and Y sex chromosomes (Groenen et al. 2012; Prather 2013). Genetic change in pigs was traditionally accomplished by selective breeding, which preferentially retained specific segments of the genome over many generations. Selection programs have dramatically changed the pig over the last century; the most notable are the long, lean, muscular, highly efficient, and very prolific attributes of today’s modern pigs. Breeding programs have altered the pig’s genome by selecting among existing genetic variation, or for new modifications that arise naturally within populations.

Genetic engineering appends and refines existing genetic variation. For example, novel genes can be added to a genome, whereas less-desirable genetics can be removed. The first intentional genetic modifications introduced into the pig were transgenes (Hammer et al. 1985). Later, homologous recombination, which results in the modification of a genetic locus, was adapted for use in the pig, leading to the first porcine gene knock-outs (Dai et al. 2002; Lai et al. 2002) and knock-ins (Rogers et al. 2008b). Gene knock-out involves disruption of a specific, pre-determined genetic sequence, resulting in the failed production of the gene’s encoded protein, whereas gene knock-in introduces a modification that can alter the coding region of a gene such that the gene’s encoded protein is altered, but is still produced; most genetic modifications are a variation on these two themes. The novelty is therefore not in the result, but rather the improvement in the tools that make these genetic changes possible.

Below, we trace the evolution of genetic engineering, from transgenes and homologous recombination to the newest, current tools available to make these changes – with particular emphasis on applications in the pig. The discussion also incorporates selected historical references that highlight the milestone discoveries that led to these tools..

Transgenes

A simple form of genetic engineering involves introducing a DNA sequence that encodes for a protein of interest. The protein-encoded sequences are generally referred to as transgenes because the DNA originates from an exogenous source. The standard, reliable technique for adding a transgene was by pronuclear injection, which was pioneered in mice (Gordon et al. 1980) and subsequently applied to other mammals such as the pig (Hammer et al. 1985). Pronuclear injection provided many options for genetic engineering since very large constructs could be injected and integrated. Thus, the coding region plus relatively long DNA segments containing regulatory regions could be introduced simultaneously.

One aspect of pronuclear injection that can be viewed as an advantage or a disadvantage is the fact that genome integration occurs randomly. Thus, the construct can integrate into a region that results in zero, low, medium, or high expression. While the consequences of random integration may be a disadvantage, in that you can’t control expression, an advantage is that the range of expression within a population of individuals should cover the level that is needed to evaluate the function of this transgene. Animals with the appropriate level of expression can then be selected and propagated (Kerr et al. 2001).

Pronuclear injection can also result in mosaic or chimeric animals. Mosaicism likely results from integration occurring after the zygotic stage. If, for example, the transgene integrates into only one nucleus of a 2-cell stage embryo, then the resulting animal will likely contain cells with and without the transgene. In fact, integration may not readily occur until the 4-cell stage or beyond in the pig (Wall and Seidel 1992) since integration is more likely to occur in genes actively undergoing transcription (Bishop and Smith 1989), and embryonic genome activation becomes highly active at the 4-cell stage (Jarrell et al. 1991). Indeed, mosaicism is less of a problem in the mouse, probably because its genome activation occurs at the 2-cell stage (Levey and Brinster 1978; Levey et al. 1978), whereas mosaicism is more prevalent in cattle, consistent with its genome activation occurring during the 8- to 16-cell stage (Frei et al. 1989).

Another concern with random integration of a transgene is that integration may occur at multiple loci (i.e. on separate chromosomes) or multiple copies can integrate at a single location. Each of the events can be viewed as an advantage that increases variation from which to select the appropriate expression level, but can also create complications when establishing genotyping protocols.

Techniques other than pronuclear injection also result in random integration, and have been successfully applied to generate transgenic pigs, including sperm-mediated gene transfer (Lavitrano et al. 1997), retroviral-mediated transduction of oocytes (Cabot et al. 2001), retroviral transduction of zygotes (Kostic et al. 2013; Whitelaw et al. 2004), other mobile genetic elements (Carlson et al. 2011), and adeno-associated virus transduction (Desauliners et al. 2016). Sperm-mediated transfection or virus-dependent transduction are even easier than pronuclear injection since an elaborate microscope and micromanipulator system or tissue culture incubators are not needed.

One way to predict the expression profile of a transgene prior to creating the animal is to introduce the transgene into somatic cells, and then to select clonal cells for appropriate expression. The cells that correctly express the transgenes can then be used for somatic cell nuclear transfer to create the transgenic animal (Park et al. 2001).

Homologous Recombination

Homologous recombination, as the name implies, relies on sequence similarity to promote chromosome targeting vector pairing so that recombination will insert a sequence of interest at a specific site (Doetschman et al. 1987). A specific chromosome location is targeted using a stretch of DNA – e.g. the “targeting vector” – that is homologous to or shares sequence with the desired site. Interspersed or localized within the targeting vector is the desired genetic change, which can include, but is not limited to, stop codons to disrupt a gene, single-base changes to alter a single amino acid, or replacement of entire exons that replace a domain in a protein. A selectable marker, such as a gene that encodes an enzyme to make the cells resistant to a drug or antibiotic, can also be included within the targeting vector. (Homologous recombination can be a rare event, so inclusion of the selectable marker provides a means to enrich for only the cells that integrated the targeting vector at a functional locus.) Genetic modifications are then transferred into animals by using the nuclei carrying the recombined DNA sequence as a donor for somatic cell nuclear transfer.

The first gene knockouts in pigs were made using homologous recombination, although the efficiency of this process was quite low – at approximately 1×10−7 (Dai et al. 2002; Lai et al. 2002). The first studies used the promoter-trap method, a technique that is dependent on an active gene’s promoter driving expression of the selectable marker, although homologous recombination has also been accomplished without selection-marker enrichment (Mendicino et al. 2011; Ramsoondar et al. 2011). The low efficiency of homologous recombination is offset by the promise that the resultant animals carry the intended genetic modification applications. Due to the high cost of such an inefficient approach, however, most published examples of homologous recombination in pigs were directed towards biomedical models (Flisikowska et al. 2012; Lorson et al. 2011; Rogers et al. 2008a).

Gene Editing

The efficiency of gene targeting can be improved by using gene editors, such as zinc finger nuclease (ZFN), Tal effector nuclease (TALEN), and the Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Each of these editing agents is equally effective at generating the desired genetic change.

ZFNs and TALENs are constructed in modular form using protein domains that recognize specific nucleotides or strings of nucleotides. ZFNs and TALENs also rely on a nuclease that must dimerize to cut the DNA, so each monomer nuclease (generally Fok1) from pairs of zinc fingers or Tal effectors need to be spatially aligned over the desired cut site. Once the DNA strands are cut, repair is initiated by non-homologous end joining – which is imperfect, so errors can be introduced. If the repair occurs correctly, then the site is thought to be repeatedly cut until the introduction of an error that prevents one or both of the ZFN or TALEN pairs from binding that region of DNA, thereby obstructing dimerization of the nuclease. Insertion or deletion of a single or a pair of bases results in a frame-shift, thus generating a gene knockout. If three bases are inserted, then a single amino acid will be inserted so long as they do not encode a stop codon.

ZFNs have been used in pigs to knock out transgenes (e.g. enhanced Green fluorescent protein [eGFP] (Whyte et al. 2011)) and endogenous genes (Glycoprotein, alpha-galactosyltransferase 1) in somatic cells, followed by somatic cell nuclear transfer, but also after injection into zygotes (Lillico et al. 2013). Similarly, TALEN have been used in both somatic cells (Carlson et al. 2012) and zygotes (Lillico et al. 2013).

In contrast to ZFN and TALEN, the CRISPR/Cas9 system relies on a single nuclease (Cas9) and a nucleotide guide sequence that is homologous to the targeted region of the chromosome. The advantage that the CRISPR/Cas9 system has over the other gene editors is the ease of construction. Guides to target the chromosomal location can be designed and constructed in a few days. In pigs, the use of the CRISPR/Cas9 was first described by Hai et al. for in vivo-produced zygotes (Hai et al. 2014), and then in somatic cells and zygotes to knock out CD1D and Cluster of differentiation 163 (CD163) using in vitro-derived eggs and zygotes (Whitworth et al. 2014). Since that time, our group has knocked out 8 other genes by zygote injection of the CRISPR/Cas9 components. In many cases, all the pigs produced had an edit at the target site: In total 83 piglets derived from zygote injection targeting 10 different genes were produced, and 62 (75%) of the piglets were edited.

These gene editors systems are not just good for knocking out a gene. If donor DNA, such as a targeting vector, is included with the gene editors, endogenous DNA repair machinery can use the donor DNA to repair the cut generated by the nucleases, resulting in “homology-directed repair”. The repair can be a single nucleotide change, or swapping of an entire exon (Whitworth et al. 2014). The option to knock in or knock out genetic sequences with gene editors provides unlimited applications of this technology for both agriculture and biomedicine (Figure 1).

Figure 1.

Figure 1

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Application of genetic engineering to pigs – whether through transgenes, homologous recombination, or introgressed edits – has application in production agriculture and medicine. Agricultural applications include answering basic questions about gene expression and protein structure, and/or basic mechanisms of cells and gene function. Practical applications include making animals resistant to disease, improving carcass composition, decreasing input requirements, and creating animals that can host the genetics of an animal of higher genetic merit. The consequences of these genetic changes may include decreasing production costs, keeping agriculture sustainable, diminishing threats to our food supply, and improving animal welfare. In some cases, the pig is also the ideal model of human disease because it permits invasive study as well as the testing of treatments and therapies. Finally, genetically engineered pigs may provide a source of reagents, e.g. tissues for xenotransplantation to humans and for pharmaceuticals (Yao et al. 2016).

Applications to Reproduction

Genetic engineering can be used to better understand various aspects of reproduction as applied to humans. Many aspects of pig reproduction are suitable as a translational model of human reproduction, including centriole inheritance, oocyte maturation, fertilization, tubo-uterine contractility, early embryo development, pregnancy and fetal programming, and reproductive disease (recently reviewed by (Mordhorst and Prather 2017). One application exemplifying what genetic engineering can teach us about pig and human reproduction is the simple introduction of a mouse Pou5f1 (also known as Oct4) promoter driving eGFP (Nowak-Imialek et al. 2011), which is expressed in pluripotent cells – albeit Pou5f1 expression in the mouse and pig is distinct (Kirchhof et al. 2000). Another application is to fuse a protein of interest to the eGFP to provide a live-cell marker that could be tracked in all cells that express eGFP under a promoter of interest. This approach was used with Proteasome subunit alpha 1, a component of the proteasome present on the sperm surface, allowing the movement of individual porcine sperm to be traced by imaging eGFP (Miles et al. 2013). These sperm are useful for evaluating the role of the proteasome during fertilization and anywhere else the ubiquitin-proteasome system functions within the female reproductive tract.

While many genetically engineered pig models of human disease are justified, there are other applications for agriculture that may not have a direct effect on human medicine, such as the expression of genes that are unique to pigs. For example, Gonadotropin-releasing hormone has two receptors in pigs (GNRHR1 and GNRHR2), whereas humans only make GNRHR1 – human GNRHR2 is a pseudogene. In the pig, GNRHR2 is ubiquitously expressed and controls luteinizing hormone-independent secretion of testosterone in the testis (Brauer et al. 2016; Desaulniers et al. 2015). Indeed, Desaulniers et al knocked down GNRHR2, and demonstrated a vital role for GNRH2 and its receptor, GNRHR2, in the production of testosterone by Leydig cells (Desauliners et al. 2016). Another gene expressed uniquely in the pig is Interleukin 1 beta 2 (IL1B2), whose expression is restricted to the peri-implantation conceptus (Mathew et al. 2015). A paradigm that allows us to address the role of IL1B2 during elongation and maternal recognition of pregnancy required creating IL1B2-knockout embryos. One strategy to generate these embryos is via CRISPR/Cas9 injection into zygotes. While the efficiency of the edits can be very high, not all of the resulting embryos will be true knockouts. Since the pig is a litter bearing species, and some of these molecules may diffuse between conceptuses, it was important to begin with genetically identical litters. Somatic cell nuclear transfer is currently the only way to ensure that all the conceptuses are knockouts, so IL1B2 was first knocked out of fetal derived fibroblast cells, whose nuclei could then be used as donors for somatic cell nuclear transfer to create a litter of embryos that were subsequently transferred to a surrogate. While we are still collecting the data, expression of IL1B2 appears to be required for conceptus elongation (see Geisert et al. 2017). Yet, many questions still remain, including: Does IL1B2 signal for maternal recognition of pregnancy? And can a few wild-type embryos in the litter provide sufficient IL1B2 signal for the IL1B2-knockout embryos to elongate? Genetic engineering technologies can therefore be used to determine the role of many other genes thought to be involved in this critical window of maternal recognition and conceptus elongation.

A tangential, but still important, application of gene-editing technology is the creation of host animals that can drive the production of gametes from germ cells derived from another individual. The first example of such a pig model involved editing NANOS2 to create a knockout. Boars with this modification lack a germ line (Park et al. 2017). The mono-allelically edited males and females are fertile, however, so these can be mated to create males that are null for NANOS2. These NANOS2-null males may provide a suitable environment to host the germ cells from a genetically superior male, and thus expanding his genetic potential.

Beyond studying the role(s) of gene(s) during gametogenesis, fertilization, development, and maternal recognition of pregnancy, genetic engineering can also be used to prevent reproductive disease. Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is estimated to cost producers in North America and Europe $6,000,000 per day (see Whitworth and Prather, 2017). The molecule implicated as a gatekeeper for viral infection is CD163; indeed, CD163-knockout pigs are resistant to PRRSV infection (Whitworth et al. 2016). Gene editing also helped implicate domain 5 of CD163 as the critical component of infection (Burkard et al. 2017; Wells et al. 2017; Whitworth and Prather 2017).

Conclusion

Techniques for genetic engineering pigs have matured to the point that most any type of genetic modification imaginable can be created in a shorter time frame and less expensively than before. The field began with the ability to add transgenes, progressed to knocking out genes, and can now modify a few select nucleotides at a time (Lillico et al. 2016). Genetically engineered pigs will soon provide a better understanding of reproductive biology, and will allow us to create animals that are more efficient, healthier to consume, and are resistant to disease. As evidence of this, the National Swine Resource and Research Center at the University of Missouri (http://nsrrc.missouri.edu) is funded to create models that have a biomedical application, and currently has over 60 strains available for distribution, with more to come soon.

Highlight.

“Genetically engineered pigs will soon provide a better understanding of reproductive biology, and allow us to create animals that are more efficient, healthier to consume, and are resistant to disease.”

Acknowledgments

The authors acknowledge funding from Food for the 21st Century and the National Institutes of Health U42 OD011140.

Abbreviations

CRISPR

clustered regularly interspaced short palindromic repeats

eGFP

enhanced green fluorescent protein

TALEN

transcription activator like effector nuclease

ZFN

zinc finger nuclease

Footnotes

The authors declare no conflict of interest.

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