Genetic Engineering Of Livestock Research Articles

The Complex and Diverse Genetic Architecture of the Absence of Horns (Polledness) in Domestic Ruminants, including Goats and Sheep.

Horns are the most obvious common feature of Bovidae. The naturally occurring absence of horns in these species, also known as polledness, is of surprisingly heterogeneous nature, although they are Mendelian traits. This review compares in detail the molecular differences among the causes of inherited polledness in the domestic ruminant species of cattle, yak, sheep, and goat based on the causal gene variants that have been discovered in recent years. The genetic causes for the lack of horns in small ruminants seem not only to be more complex, e.g., in sheep, breed-specific characteristics are still unexplained, but in goats, there is also the associated disorder of intersexuality—polled intersex syndrome (PIS). In connection with animal welfare and the associated discussion about a legal ban on the dehorning of all farm animals, naturally hornless animals and the causal genetic variants are of increasing research interest in the age of genome editing. However, the low acceptance of genetic engineering in livestock, especially in European societies, limits its use in food-producing animals. Therefore, genotype-based targeted selection of naturally occurring variants is still a widely used method for spreading this desired trait within and across populations, at least in cattle and sheep.

Genetic Engineering of Livestock: The Opportunity Cost of Regulatory Delay.

Genetically engineered (GE) livestock were first reported in 1985, and yet only a single GE food animal, the fast-growing AquAdvantage salmon, has been commercialized. There are myriad interconnected reasons for the slow progress in this once-promising field, including technical issues, the structure of livestock industries, lack of public research funding and investment, regulatory obstacles, and concern about public opinion. This review focuses on GE livestock that have been produced and documents the difficulties that researchers and developers have encountered en route. Additionally, the costs associated with delayed commercialization of GE livestock were modeled using three case studies: GE mastitis-resistant dairy cattle, genome-edited porcine reproductive and respiratory syndrome virus-resistant pigs, and the AquAdvantage salmon. Delays of 5 or 10 years in the commercialization of GE livestock beyond the normative 10-year GE product evaluation period were associated with billions of dollars in opportunity costs and reduced global food security.

Current progress of genome editing in livestock

Historically, genetic engineering in livestock proved to be challenging. Without stable embryonic stem cell lines to utilize, somatic cell nuclear transfer (SCNT) had to be employed to produce many of the genetically engineered (GE) livestock models. Through the genetic engineering of somatic cells followed by SCNT, GE livestock models could be generated carrying site-specific modifications. Although successful, only a few GE livestock models were generated because of low efficiency and associated birth defects. Recently, there have been major strides in the development of genome editing tools: Zinc-Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENS), and Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (Cas9) system. These tools rely on the generation of a double strand DNA break, followed by one of two repair pathways: non-homologous end joining (NHEJ) or homology directed repair (HDR). Compared to the traditional approaches, these tools dramatically reduce time and effort needed to establish a GE animal. Another benefit of utilizing genome editing tools is the application of direct injection into developing embryos to induce targeted mutations, therefore, eliminating side effects associated with SCNT. Emerging technological advancements of genome editing systems have dramatically improved efficiency to generate GE livestock models for both biomedical and agricultural purposes. Although the efficiency of genome editing tools has revolutionized GE livestock production, improvements for safe and consistent application are desired. This review will provide an overview of genome editing techniques, as well as examples of GE livestock models for agricultural and biomedical purposes.

CRISPR/Cas9-mediated MSTN disruption and heritable mutagenesis in goats causes increased body mass.

Genetic engineering in livestock has been greatly enhanced through the use of artificial programmed nucleases such as the recently emerged clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. We recently reported our successful application of the CRISPR/Cas9 system to engineer the goat genome through micro-injection of Cas9 mRNA and sgRNAs targeting MSTN and FGF5 in goat embryos. The phenotypes induced by edited loss-of-function mutations of MSTN remain to be evaluated extensively. We demonstrate the utility of this approach by disrupting MSTN, resulting in enhanced body weight and larger muscle fiber size in Cas9-mediated gene-modified goats. The effects of genome modifications were further characterized by H&E staining, quantitative PCR, Western blotting and immunofluorescence staining. Morphological and genetic analyses indicated the occurrence of phenotypic and genotypic modifications. We further provide sufficient evidence, including breeding data, to demonstrate the transmission of the knockout alleles through the germline. By phenotypic and genotypic characterization, we demonstrated the merit of using the CRISPR/Cas9 approach for establishing genetically modified livestock with an enhanced production trait.

Genome editing and genetic engineering in livestock for advancing agricultural and biomedical applications.

Genetic modification of livestock has a longstanding and successful history, starting with domestication several thousand years ago. Modern animal breeding strategies predominantly based on marker-assisted and genomic selection, artificial insemination, and embryo transfer have led to significant improvement in the performance of domestic animals, and are the basis for regular supply of high quality animal derived food. However, the current strategy of breeding animals over multiple generations to introduce novel traits is not realistic in responding to the unprecedented challenges such as changing climate, pandemic diseases, and feeding an anticipated 3billion increase in global population in the next three decades. Consequently, sophisticated genetic modifications that allow for seamless introgression of novel alleles or traits and introduction of precise modifications without affecting the overall genetic merit of the animal are required for addressing these pressing challenges. The requirement for precise modifications is especially important in the context of modeling human diseases for the development of therapeutic interventions. The animal science community envisions the genome editors as essential tools in addressing these critical priorities in agriculture and biomedicine, and for advancing livestock genetic engineering for agriculture, biomedical as well as "dual purpose" applications.

Advances in genetic engineering of domestic animals

Global population will increase to over nine billion by 2050 with the doubling in demand for meat and milk. To overcome this challenge, it is necessary to breed highly efficient and productive livestock. Furthermore, livestock are also excellent models for human diseases and ideal bioreactors to produce pharmaceutical proteins. Thus, genetic engineering of domestic animals presents a critical and valuable tool to address these agricultural and biomedical applications. Overall, genetic engineering has evolved through three stages in history: transgenesis, gene targeting, and gene editing. Since the birth of the first transgenic pig, genetic engineering in livestock has been advancing slowly due to inherent technical limitations. A major breakthrough has been the advent of somatic cell nuclear transfer, which, for the first time, provided the technical ability to produce site-specific genome-modified domestic animals. However, the low efficiency of gene targeting events in somatic cells prohibits its wide use in agricultural and biomedical applications. Recently, rapid progress in tools and methods of genome engineering has been made, allowing genetic editing from mutation of a single base pair to the deletion of entire chromosomes. Here, we review the major advances of genetic engineer- ing in domestic animals with emphasis placed on the introduction of latest designer nucleases.

Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs.

Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs.

VIRAL AND NONVIRAL VECTORS

Genetically engineered (GE) livestock have existed since the mid-1980s, and, since then, a range of methods for delivery of the transgene have been developed, each with advantages and limitations. In regards to the wealth of possible methods for the production of GE animals, two general approaches have emerged. Historically first, the direct manipulation of the zygote (including manipulation of germ cells) now comes in many flavours, from direct pronuclear injection of double-stranded DNA constructs to the use of vectors to deliver the transgene. The second approach utilises an in vitro stage where cells are engineered in culture before being introduced in some way to the developing embryo. The former suffers from lack of control of transgene integration, which can expose the transgene to position effects. The latter can be exploited through homologous recombination to engineer specific genetic loci; with somatic cell cloning being the most widely used method. Both approaches can now be combined with the exciting new editor technologies to enable precise genome editing which in some cases does not involve the incorporation of a transgene. For methods involving the zygote, the use of specific vectors can be of advantage, and the same can be true for the manipulation of cells; however, many delivery strategies are possible for this process. Overall, the drivers for delivery method development have revolved around efficiency and specificity. With regard to viral vectors, and possibly nonviral nanoparticle formulations in the future, spectacular increases in transgenesis rates can be achieved. With the most widely used vector, based on a lentivirus genome, in some cases all animals born from injected zygotes can be transgenic. In livestock, where gestation and breeding times are long, this dramatically reduces the time to proof-of-concept for a given project. In addition, these founder animals will carry different transgene copy-numbers, which is associated with different levels of transgene expression. This strategy can be exploited to quickly produce a large cohort of animals that enable modelling of the range of phenotype observed in a population for a given disease. In addition to the delivery of a transgene, such vectors can also be beneficial for the delivery of reagents that facilitate genome engineering, the most exciting of which are the genome editors. In this situation, either the editor and/or any DNA sequence to be incorporated can be efficiently delivered if not essential for broad uptake of this approach. It is likely that nonintegrating vectors will be desirable. In summary, viral vectors have a broad utility in facilitating the production of GE animals. In the future, nonviral nanoparticles may offer similar opportunities. Given the breadth of methodologies available and with the anticipated use of GE livestock in both agricultural and biomedical applications gaining momentum, we are entering an era of unparalleled opportunity in this area of animal biotechnology.

THE QUEST FOR PORCINE PLURIPOTENT STEM CELLS

The isolation of embryonic stem cells (ESC) and embryonic germ cells (EGC) from early embryos is a major milestone in modern science and holds a great potential for human medicine. In 2007, Shinia Yamanaka and co-workers reprogrammed somatic cells to pluripotency by induced expression of pluripotency transcription factors. These so-called induced pluripotent stem cells (iPSC) are equivalent to ESC in terms of pluripotency and have the same potential for use in regenerative therapies. However, before the use of pluripotent cells or their derivatives in humans, potential therapies need to be tested in suitable animal models to ensure their safety. In this respect, the domestic pig is particularly suited for the testing of stem cell-based therapies intended for humans, since in general physiology and metabolism are similar in human and pigs. Since the isolation of the different types of pluripotent cells in human and mouse, there have been reports of derivation of ESC-like and EGC-like cell lines from porcine embryos. Despite the significant progress that has been reported in these studies, none of the described porcine cell lines have fulfilled all of the criteria for pluripotency, such as long-term maintenance and the ability to differentiate into all of the cells in the organism, including the germ line. This has prevented the use of these cells in the genetic engineering of livestock as well as their therapeutic application in animal models for human diseases. The derivation of the first porcine cell lines with iPSC characteristics (Ezashi et al. 2009 PNAS 27, 10 993–10 998) has provided a viable alternative to the ESC/EGC, and some major successes have been already achieved. The majority of the putative iPSC described in the literature have demonstrated pluripotent characteristics such as expression of various pluripotency markers and an ability to differentiate into the three primary germ layers in vivo by forming teratomas in immunodeficient mice. One group has reported the derivation of iPSC lines that have been capable to generate chimeras with germline contribution (West et al. 2011 Stem Cells 29, 1640–1643), which is the first fully confirmed report of successfully produced porcine germ line chimera to date. Additionally, the differentiation of putative iPSC into rod photoreceptors and their integration into the retinas of recipient pigs has been reported (Zhou et al. 2011 Stem Cells 29, 972–980). Despite these major achievements, some challenges remain to be overcome in order to make porcine iPSC more widely applicable in disease models and in the transgenic technology. Due to some variations in the morphological and molecular characteristics of the reported putative iPSC lines, it needs to be determined which markers are the hallmarks of truly pluripotent porcine iPSC. Second, it is still not clear which are the optimal culture conditions for derivation and long-term culture of these cells. Since the culture conditions used today have been proven ineffective to maintain pluripotency in porcine ESC and EGC, the question remains whether the continuous expression of the transgenes is an important factor in the long-term culture of iPSC. Finally, it needs to be determined whether putative porcine iPSC derived from cell types other than multipotent stem cells (such as mesenchymal stem cells used by West et al., 2011) possess full pluripotency, which should be demonstrated by germ line chimera production via blastocyst injection or tetraploid complementation.

Applications of RNA interference-based gene silencing in animal agriculture

Classical genetic selection, recently aided by genomic selection tools, has been successful in achieving remarkable progress in livestock improvement. However, genetic selection has led to decreased genetic diversity and, in some cases, acquisition of undesirable traits. In order to meet the increased demands of our expanding population, new technologies and practices must be developed that contend with zoonotic and animal disease, environmental impacts of large farming operations and the increased food and fibre production needed to feed and clothe our society. Future increases in productivity may be dependent upon the acquisition of genetic traits not currently encoded by the genomes of animals used in standard agricultural practice, thus making classical genetic selection impossible. Genetic engineering of livestock is commonly used to produce pharmaceuticals or to impart enhanced production characteristics to animals, but has also demonstrated its usefulness in producing animals with disease resistance. However, significant challenges remain because it has been more difficult to produce animals in which specific genes have been removed. It is now possible to modify livestock genomes to block expression of endogenous and exogenous genes (such as those expressed following virus infection). In the present review, we discuss mechanisms of silencing gene expression via the biology of RNA interference (RNAi), the technology of activating the RNAi pathway and the application of this technology to enhance livestock production through increased production efficiency and prevention of disease. An increased demand for sustainable food production is at the forefront of scientific challenges and RNAi technology will undoubtedly play a key role.

Embryonic stem cells in fish: current status and perspectives.

Totipotent embryonic stem (ES) cells represent a bridge that links in vitro and in vivo manipulations of animal genomes and have enormous potential for genetic engineering of livestock. We have recently established feeder cell-free conditions for culturing cells of midblastula embryos (MBE) of the medaka (Oryzias latipes) and obtained several stable cell lines that show all features of mouse ES cells in vitro. One of these lines, MES1, has been demonstrated to retain a diploid karyotype and can be induced to differentiate into various cell types in vitro. Upon microinjection into albino host blastulae, MES1 cells are able to form pigmented chimeras. Genotype-specific PCR analysis revealed that 90% of host blastulae transplanted with MES1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. Transplantation of genetically labelled MES1 cells revealed a wide contribution to numerous organs derived from all three germ layers and differentiation into various types of functional cells. These ES properties of MES1 line was not abolished by stable gene transfer and long-term selection. Thus MES1 cells may represent a first promising cellular vehicle for the production of genetically modified fish. The genetic background has been found to have a profound effect on the efficacy of ES cell derivation and of chimera formation.

Transgenic domestic animals provide an animal model for rheumatoid arthritis

Production of transgenic domestic animals by microinjection of egg nuclei has permitted the introduction of exogenous genes into the transgenic animal. Pursell et al (1) report severe synovitis, cardiomegaly, dermatitis, renal disease and gastric ulcers in pigs with an expressed bovine growth hormone transgene. I have hypothesized that rheumatoid arthritis (RA) is a disease with initial symptoms to the gamete (2, 3). This hypothesis is supported by data showing a reduced rate of RA in women using oral contraceptives (4, 5) and remission of RA in pregnancy (6). RA in animals resulting from trauma to the gamete would be consistent with this hypothesis. This paper hypothesizes that some of the pathology associated with genetic engineering of livestock can be attributed to a rheumatoid arthritis-like spectrum of symptoms. This systemic reaction could be sufficiently similar to human RA to permit the transgenic pig to serve as an animal model of the disease.

Genetic Engineering of Livestock

Genetic engineering of livestock is expected to have a major effect on the agricultural industry. However, accurate assessment of the consequences of transgene expression is impossible without multigenerational studies. A systematic study of the beneficial and adverse consequences of long-term elevations in the plasma levels of bovine growth hormone (bGH) was conducted on two lines of transgenic pigs. Two successive generations of pigs expressing the bGH gene showed significant improvements in both daily weight gain and feed efficiency and exhibited changes in carcass composition that included a marked reduction in subcutaneous fat. However, long-term elevation of bGH was generally detrimental to health: the pigs had a high incidence of gastric ulcers, arthritis, cardiomegaly, dermatitis, and renal disease. The ability to produce pigs exhibiting only the beneficial, growth-promoting effects of growth hormone by a transgenic approach may require better control of transgene expression, a different genetic background, or a modified husbandry regimen.