Why sheep

Their rectangular pupils allow them to have a to degree field of vision. This is especially useful for sheep as they are a prey species and need to always be on watch for predators. A study revealed that sheep can recognize up to 50 other sheep faces, and remember them for two years.

In this study, sheep learned to distinguish between 25 pairs of sheep by associating one sheep from each pair with a food reward. The sheep were tested at various time intervals afterwards, and it was found that sheep could remember the faces that lead to a food reward up to two years later. Other studies have shown they are capable of learning and remembering how to navigate complex mazes. They use plants and other substances that otherwise hold no nutritional value to them to prevent or treat disease, and teach their young to do the same.

Sheep are capable of experiencing a wide range of emotions, just as humans do. Many studies have highlighted the ability for sheep to feel afraid, angry, bored, sad and happy. Not only are sheep able to feel basic emotions, but their feelings can be very complex and include interactions between emotions and cognition. For example, believe it or not, scientific studies have shown that sheep can be pessimists and optimists! This groove is called a philtrum.

Many ethnic customers prefer to perform their own slaughter in accordance with their cultural and religious beliefs. The two primary religions that practice ritual slaughter are Jews Kosher and Muslims Halal. Though the Humane Slaughter Act requires that animals be rendered insensible prior to their slaughter, religious or ritual slaughter is exempted from the Humane Slaughter Act.

Animals killed for halal or kosher, according to religious law, cannot be stunned or rendered unconscious before their throats are cut. Instead, the jugular veins, carotid arteries, esophagus, and trachea of the animal are severed with a deep cut while the animal is restrained and fully conscious—which means that many animals—like the cow shown in this video —fight and gasp for their last breath, struggling to stand while the blood drains from their necks.

From winter coats to decorative rugs, to delicious stews and curries, it is possible to enjoy traditional recipes and well-made products without the traditional cruelty. Cotton, cotton flannel, polyester fleece, synthetic shearling, acrylic and hemp are all alternatives to wool that are cheaper and easier to care for.

And please visit the Recipe Roundup on our Vegan Nutrition page, for links and ideas to delectable animal-friendly cooking. Stay at The Gray Barn! Help farmed animals: join our email list! Sheep for Meat and Wool To my mind, the life of a lamb is no less precious than that of a human being.

Mahatma Gandhi Sheep are intelligent, inquisitive animals with good memories. Painful Mutilation Sheep raised for wool and meat also face a variety of painful mutilations. Slaughter of Babies In addition to the sale of wool, the other main source of income from sheep production is the sale of lambs for meat.

Make a donation. Farm animals Sheep Read more. Read more. Sheep welfare The main welfare issues affecting sheep are the result of mutilations, lameness, transport, and illness caused by disease. Sign up for our newsletter. Keep informed about urgent actions and other ways to help. Sign Up.

Our Christmas cards featuring beautiful artworks by Rachel McNaughton are available now. Available to order now. Gene-edited sheep and goats, generated using these tools, provide valuable models for investigations on gene functions, improving animal breeding, producing pharmaceuticals in milk, improving animal disease resistance, recapitulating human diseases, and providing hosts for the growth of human organs. In addition, more promising derivative tools of CRISPR systems have emerged such as base editors which enable the induction of single-base alterations without any requirements for homology-directed repair or DNA donor.

These precise editors are helpful for revealing desirable phenotypes and correcting genetic diseases controlled by single bases.

Generating new and variable phenotypes via direct alteration of DNA sequences is an interesting idea that has sparked the curiosity of a wide spectrum of researchers over the past few decades. Based on significant efforts, tremendous advances have been achieved in animal genetics and reproductive physiology. These have enabled what is now known as the genome-editing revolution that can be applied to generate gene-edited animals including sheep and goats for various purposes Figure 1.

Figure 1 Applications and aims of genome engineering in sheep and goats. Genome engineering has been applied in both sheep and goats or generally in farm animals for various purposes such as to investigate the biological and functional roles of genes, to introduce novel economically important traits for agricultural purposes, to produce valuable proteins in milk, to produce animals that are resistant to epidemic diseases, to model human diseases, and to produce hosts for the growth of human organs for xenotransplantation research, among other valuable purposes that mainly aim to increase human knowledge, as well as human and animal health and welfare.

About 40 years ago, a set of basic techniques were applied to sheep embryos with the desire to generate identical twins, multiplets, and chimeras. The further development of these tools has led to the generation of identical individuals by embryo splitting Willadsen, , chimeras by aggregating embryonic cells Fehilly et al. During that time, in , the first report about the generation of transgenic farm animals including sheep via pronuclear injection PNI was published, announcing the first procedure for the production of transgenic farm animals Hammer et al.

About 10 years later, in , success of cloning sheep from more differentiated embryonic cells has been reported Campbell et al. One year later, the same group announced unprecedented success by cloning the sheep Dolly from adult somatic cells Wilmut et al. In the same year, another remarkable advance had been achieved by using transfected fetal fibroblast cells for the generation of the first transgenic cloned sheep Schnieke et al. Based on these advances, somatic cell nuclear transfer SCNT has been established as an essential tool for the creation of transgenic animals.

From that time, various strategies have been applied to facilitate the generation of gene-modified animals that express specific and desired traits, employing spermatozoa, viral vectors, transposons, recombinases, RNA interference RNAi molecules, and endonucleases Figure 2. Table 1 Examples of transgenic sheep and goats produced using a pronuclear microinjection PNI approach. Table 2 Examples of transgenic and gene-targeted sheep and goats produced using a somatic cell nuclear transfer SCNT approach.

Figure 2 Schematic representation of practical and likely pathways of genetic modification in sheep and goats. Pronuclear injection PNI and nuclear transfer NT are the two primary procedures for the generation of live founders with desired genetic modifications. In addition to these two approaches, several new tools have emerged that increase the efficiency and simplify the process of mediating genetic modification. These have served to mediate manipulations in a variety of cells and organs, including somatic cells, embryonic cells, embryos, spermatozoa, spermatogonial stem cells SSCs , testes, mammary glands, and other targeted organs.

Different procedures are involved in the delivery of DNA constructs as well as the various enzymes and systems that induce genetic modification events within genomes.

PNI, cytoplasmic injection CI , perivitelline space injection PSI , and zona-free transduction ZFT have been used for the delivery to embryos, transfection TF , and transduction TD for the delivery to cells, incubation IC for the delivery to spermatozoa, intratesticular injection ITI for the delivery to testes, intramammary injection IMI for the delivery to mammary glands, and direct injection DI for the delivery to targeted organs mainly for medical purposes.

In vitro fertilization IVF , intracytoplasmic sperm injection ICSI , artificial insemination AI or even natural mating NM have been used for the delivery of transgenic sperms that resulted from incubation treatment, male germ cell transplantation, or intratesticular injection. In the diagram, from left to right, red arrows indicate the uses of DNA constructs for mediating DNA modification, green arrows indicate the uses of viral vectors, yellow arrows indicate the uses of RNAi molecules via delivery by viral vectors, purple arrows indicate the uses of transposons, pink arrows indicate the uses of RNAi molecules via integration by transposons, tan arrows indicate the independent uses of RNAi molecules, light blue arrows indicate the uses of recombinases, and orange arrows indicate the uses of endonucleases.

Isolation I of spermatogonial stem cells from transgenic males can be used via transplantation T into infertile males to generate donor-derived spermatogenesis, which can then be used to generate transgenic founders. Furthermore, isolation of cells from transgenic individuals can also be used by nuclear transfer NT to generate transgenic progeny.

Other abbreviations used in the diagram include embryo transfer ET , lactation L , and delivery D. In addition to the other toolkits of genome engineering, CRISPR systems have shown unprecedented potential for the generation of gene-edited animals with defined genetic alterations.

Menchaca et al. Tremendous advances in the field of genetic engineering in animals have been achieved over the past few decades. Various strategies have been used to generate genetically modified animals with desired traits Figures 1 and 2.

Increasing the efficiencies of mediating specific genetic modifications and simplifying the procedures for generating genetically modified organisms were the main aims that challenged specialists in this field. Enormous and collective efforts have been made in science and technology to facilitate the ability to induce specific genomic manipulations using procedures that have become more efficient and simpler to use.

In the following paragraphs, the various tools for mediating genetic manipulations in sheep and goats Figure 2 as well as some other relevant biotechnological advances are presented. Cloning by embryo splitting, also known as twinning by separation of blastomeres, is a set of primary reproductive techniques that have been applied to fulfill purposes related to the production of identical twins, multiplets, and chimeras.

Embryo-splitting techniques were among the first strategies that led to the emergence of cloning. Based on the concept of cloning, researchers could produce genetically modified sheep and goats by using SCNT. Therefore, these multiplication strategies are discussed in this review. Previous work on sea urchins, salamanders, rats, rabbits, and mice have led to the application of these techniques in sheep and goats Seidel, ; McLaren, ; Vajta and Gjerris, Sheep were among the first domestic animals that are subjected to embryo-splitting techniques.

In , Willadsen introduced a simple and successful procedure for blastomere separation in sheep Willadsen, Willadsen aimed to develop new and highly selective methods for the breeding of farm animals, in addition to utilizing cells of cleaved embryos to increase the number of available embryos from superior mothers for the production of valuable offspring.

Basically, the established procedure included the collection of cleaved embryos from super-ovulated donor females, blastomere separation, placement of each half into the zona pellucida, embedding in agar, and finally, transfer to recipient mothers. Embryo splitting was also applied to other livestock species including goats for both experimental and commercial purposes Tsunoda et al. Different strategies have been adapted to improve the efficiency to produce an increased number of monozygotic animals.

These strategies include the evaluation of isolated cells from different stages of development Willadsen, ; Willadsen, , presence or absence of intermediate hosts Gatica et al. The production of monozygotic animals via embryo splitting was a useful procedure for research and study of embryo development; however, the application of this procedure remained limited.

Moreover, technical difficulties and suboptimal pregnancy rates lead to the production of only a relatively small number of individuals using this procedure. This is due to the limited divisibility of embryos to obtain two or occasionally up to four genetically identical animals.

Although blastomere separation is considered as one of the basic cloning approaches, more promising approaches such as nuclear transplantation have opened the way for the large-scale production of genetically identical individuals. Despite the family relationship between sheep and goats, hybridization between both genera is an extremely rare event.

By using an embryo aggregation strategy, researchers were able to produce sheep—sheep intraspecific chimeras Tucker et al. These fundamental studies indicated the aggregation ability of isolated blastomeres from early cleavage stage embryos to produce chimeric blastocysts that can be transferred to foster mothers recipients for the production of intraspecific chimeras.

Based on these experiences and with the aim to increase the understanding of reproductive incompatibilities between species, in addition to providing a successful approach for interspecific hybridization, researchers were able to generate sheep—goat chimeras by using two basic techniques: embryo aggregation Meinecke-Tillmann and Meinecke, ; Fehilly et al.

These procedures were based on the combination of blastomeres of two species, surrounding blastomeres of two species with each other, or injecting cells of the inner cell mass of one species into the blastocyst cavity of different species. Sheep—goat chimeras differ from sheep—goat hybrids, which can be obtained when a goat naturally mates with a sheep. The phenotypic characteristics of sheep—goat chimeras include regions of both sheep-like wool and goat-like hair. Due to the mosaic nature of goat and sheep tissues in the produced chimeras, chimeric characteristics cannot be transferred to the next generation.

Fertile sheep—goat chimeras can either pass on sheep or goat characteristics to their progeny depending on whether the reproductive organs of the chimera formed from caprine or ovine origins Amoah and Gelaye, Interspecific chimerism may offer experimental approaches for developmental biology to investigate cell linkages, embryonic development interactions, reproductive incompatibilities, and embryo transfer opportunities.

Although these approaches have been used to remove the reproductive barriers between species, the expanded use of these techniques to create new hybrids remained limited. PNI was among the first techniques that have been applied to generate transgenic animals. It was the dominant methodology for the generation of transgenic animals during the first decade of animal transgenesis studies.

By introducing DNA constructs into the pronuclei of fertilized eggs and transferring the injected eggs to foster mothers, researchers were able to generate transgenic animals Figure 2.

After reporting the generation of transgenic mice using this technique Gordon et al. Sheep were among the first reported transgenic domestic animals; however, although Hammer et al. The authors speculated that the reasons behind the low efficiencies might be due to the concentration of injected DNA, the composition of the used buffer, the stage of the collected embryos, and other structural aspects of the chromosomes.

Further attempts have been made to overcome these obstacles and to facilitate the generation of transgenic animals using this technique. A few years later, in , Ebert et al. Several challenges caused these low efficiencies such as the random integration and the variable copy number of integrated DNA constructs. Because of these factors, the expression of the transgene can be unpredictable. A further technical challenge related to the application of PNI in livestock is the visualization of pronuclei.

This is obstructed by the presence of a large amount of lipid granules in livestock eggs, which results in a nontransparent cytoplasm, thus hampering the localization of pronuclei. The pronuclei of ovine eggs can be visualized using differential interference contrast DIC microscopy Hammer et al. Despite the suboptimal efficiencies of the conventional PNI, a large number of transgenic sheep and goats have been generated. Prominent examples of generated transgenic sheep and goats using PNI are shown in Table 1.

The contributions of classical PNI of DNA constructs equipped the global transgenic sheep and goat sector with novel and useful genetically modified models.

Cloning can happen naturally in a number of living organisms via asexual reproduction and can also be artificially introduced in mammals by using primary techniques such as embryo splitting Vajta and Gjerris, Efforts in embryo manipulation research led to the development of technical tools that enabled the production of identical individuals as well as intra- and inter-specific chimeric individuals Willadsen, ; Fehilly et al.

During the second wave of the development of these enabling technical tools, new and more advanced techniques emerged. Nuclear transfer, or nuclear transplantation, as it was first called, was developed to overcome the limitations of embryo-splitting techniques such as the limitation of the number of individuals that can be produced from a single split embryo. Despite the first attempts of nuclear transplantation in non-mammalian animal species and laboratory mice Meissner and Jaenisch, , the first cloned mammal sheep from undifferentiated embryonic blastomeres was reported in by Willadsen Willadsen, Willadsen used ovine 8 to cell stage embryos as nuclear donors in combination with ovine enucleated metaphase II oocytes as recipient cytoplasts to produce live lambs.

Willadsen aimed to define suitable conditions required for the large-scale cloning of domestic animals. About 10 years later, in , Campbell et al. Campbell et al. This was in response to the unsuitability of the utilization of this type of cells in genetic modification programs Colman, In the same year, a further advance has been accomplished by the generation of the first transgenic cloned sheep carrying human coagulation factor IX hFIX gene from transfected fetal fibroblasts Schnieke et al.

This success of cloning approaches in sheep was followed by several attempts to clone various species, including goats. Cloned goats were first produced by early embryonic blastomeres Yong et al. Subsequently, fetal somatic cells were used for the generation of transgenic cloned goats carrying human antithrombin III hAT gene Baguisi et al.

The aims of the cloning of domestic animals have been altered due to rapid and significant advances in the field. In addition to utilizing this approach as a valuable tool in embryological studies and to achieve the multiplication of desired genetics, nuclear transfer has become one of the basic methods to generate genetically modified animals with useful and desired traits.

Somatic cell cloning or SCNT has emerged with the creation of Dolly , the sheep from a mammary gland cell of a 6-year-old ewe, taken by Wilmut and his colleagues in Wilmut et al. In addition to the putative aims of animal cloning such as multiplying superior animals for the construction of highly productive flocks for agricultural purposes and the restoration of endangered or even extinct species, the application of SCNT in genetic modification programs of farm animals has attracted wide attention.

The emergence of SCNT has removed the barriers that inhibited the implementation of gene targeting by homologous recombination HR in species that lack embryonic stem cells ESCs to generate authentic genetically modified individuals.

Moreover, implementation of SCNT for the manipulation of animal genomes has overcome several of the drawbacks of previously emerged PNI such as the low level of transgene integration, the variability of transgene expression, the unpredictable transmission of the transgene to the next generation, and founder mosaicism.

Transgenic farm animals can be produced using SCNT via transfection of donor cell nuclei with DNA expression constructs or vectors or by cloning transgenic founder animals Figure 2.

Various cell types have been utilized as nucleic donors to generate cloned sheep and goats. These include adult mammary gland cells Wilmut et al. Fetal fibroblast cells have been used dominantly for the generation of transgenic cloned sheep and goats among other reported cell types. After the first generation of transgenic cloned sheep reported in Schnieke et al.

Despite the relatively low efficiency of SCNT and the potential for developmental anomalies, in parallel with the PNI technique, SCNT has become a basic and dominant methodology to generate transgenic and gene-targeted sheep and goats. In addition to the promising advantages of SCNT for the multiplication of genetically valuable or superior livestock and the manipulation of the genomes of experimentally, biomedically, and agriculturally important animals, SCNT offers promising potential for the conservation of genomes of endangered species and for restoring or reviving the genomes of extinct species.

Finding effective tools to conserve and restore threatened genomes is equally important to finding new tools for the manipulation of existing genomes to generate novel and desirable phenotypes. Interspecies cloning or interspecies somatic cell nuclear transfer iSCNT is one of the emerging strategies to conserve genetic diversity and prevent the rapid loss of animal genetic resources. Genetic rescue programs based on iSCNT use nuclei from endangered species in the wild, whose oocytes are difficult to obtain, with oocytes from closely related domesticated species to reconstruct embryos that can then be transferred to foster mothers.

The resultant offspring of this process resembles nucleic donors. Sheep and goats were among the closely related domesticated species that were utilized in the conservation cloning programs of threatened species that belong to the genera Ovis and Capra.

Loi et al. This provided an encouraging example of the application of iSCNT for the generation of live founders.

Further examples of the implementation of iSCNT using sheep and goats to reconstruct embryos between species within the same genera are listed in Table 4.

Despite the successful attempts of reconstructing embryos between two species from closely related genera, the number of viable offspring produced using this strategy was very low. Such low efficiency might be a result of implantation failure or of immunological rejection Wang et al. Other embryonic combinations have also been reported for the study of developmental ability, mitochondrial heteroplasmy, and nuclear-cytoplasmic interactions between different species Table 4.

Despite the low numbers of publications reporting the successful generation of viable offspring using iSCNT, research in this field still offers great potential in interspecies embryological studies and genetic resource conservation programs. Table 4 Examples of interspecies somatic cell nuclear transfer iSCNT applications in sheep and goats for the reconstruction of embryos between different species.

This modified technique has emerged to overcome the technical difficulties that obstruct the improvement and widespread application of SCNT. In traditional SCNT, oocyte enucleation is one of the technical steps which encounter major obstacles due to the presence of the zona pellucida the outer thick membrane of mammalian oocytes Lagutina et al. Thus, introducing careful manipulations inside the zona pellucida to replace the nuclei enucleating the oocyte nucleus and transferring the somatic cell nucleus requires expensive instruments such as micromanipulators as well as both skill and time Vajta and Gjerris, The simplicity of HMC is mainly based on the removal of the zona pellucida after maturation and before enucleation.

In this case, sophisticated micromanipulators are not necessary because the manipulations required for both enucleation and nucleus transfer are performed by hand as indicated by the name Vajta, Basically, the procedure of HMC includes handmade bisection of zona-free oocytes, staining and selection of cytoplasts, and fusion of the somatic cell with two cytoplasts to generate an equally sized reconstructed embryo Vajta et al.

Thus, the implementation of SCNT using this approach requires less expertise, time, and cost. Initial attempts to use zone-free procedures, especially in embryonic cell nuclear transfer, have led to the first successful report to produce cloned cattle, using a somatic cell as nucleus donor Vajta et al. In sheep and goats, initial publications have reported the application of this technique for successful embryo development Peura and Vajta, ; Akshey et al.

An interesting example of the application of this technique in sheep is the generation of transgenic cloned lambs carrying a modified nematode mfat-1 gene to enrich muscles and other organs and tissues with omega-3 fatty acids Zhang et al. Despite the relatively equivalent efficiency of HMC compared to traditional SCNT, as well as the further advantages of HMC simpler to use, cheaper, and more time-saving , the applications of HMC in sheep and goats to produce viable founders have not been studied in detail.

In general, despite the simplification provided by HMC and other emerging strategies, SCNT remains technically challenging, and few research groups around the world are able to perform it efficiently Tan et al. Further advances in this field are required to enable the widespread application of these techniques to facilitate multiplication, transgenesis, and genetic rescue of threatened genomes.

Spermatozoa have the natural ability to obtain exogenous DNA by a simple incubation procedure Brackett et al. This significant observation opened the way for further alternative strategies that can be utilized in transgenesis programs.

Basically, three main strategies have been used to mediate transgenesis that utilize the male side spermatozoon , namely, male germ cell transplantation—mediated transgenesis, sperm-mediated gene transfer SMGT , and testis-mediated gene transfer TMGT Figure 2.

In addition to its importance for spermatogenesis and fertility studies, male germ cell transplantation has been suggested to be an alternative tool to mediate transgenesis. Spermatogonial transplantation uses isolated spermatogonial stem cells SSCs from desirable male donors and injects and transplants these into the seminiferous tubules of an infertile recipient males, which results in donor-derived spermatogenesis. In this case, transgenesis can be mediated by manipulating SSCs prior to transplantation into the recipient males or by transferring transgenic donor germ cells from transgenic individuals Hill and Dobrinski, This technique has been initially established in rodents Brinster and Avarbock, ; Brinster and Zimmermann, Although this technique has basically been extended to farm animals including goats Honaramooz et al.

Homozygous knockout males showed an ablation of the male specific germline with intact testicular development, while heterologous knockout males and females were fertile. This offers an advantage in agriculture where these models serve as recipients for donor spermatogonial stem cells from genetically valuable males, thus expanding the availability of desirable genetics.

After a simple step of incubating sperm cells with exogenous DNA, transfected sperm cells can then be transferred to female eggs using various strategies, such as artificial insemination AI Zhao et al. SMGT application in sheep and goats includes the production of both transgenic embryos Pereyra-Bonnet et al. Despite the simplicity this approach offers, its application for the generation of transgenic sheep and goats remained limited.

This might be a result of a number of drawbacks of this approach such as the low incorporation of the exogenous genes. In general, several attempts have been reported to enhance the ability of sperm to obtain exogenous DNA. These include electroporation-, liker-, retroviral-, liposome lipofection -based SMGT, restriction enzyme—mediated integration, and further techniques Smith, The optimization of this approach might increase its efficiency.

TMGT or intratesticular injection are further alternative tools based on the direct injection of testes with exogenous DNA. After a specific interval, injected males can then be used to naturally mate with females to produce transgenic founders.

TMGT was initially applied in sheep and goats to produce transgenic founders with inserted genes including lipoprotein lipase LPL Qin et al. In general, despite the potential possibilities of sperm-based strategies to mediate transgenesis, these strategies still require optimization. Integration between these strategies and newly emerging targeting tools might be of great importance for the generation of desirable genetically modified sheep and goats.

Viral vectors have been used to mediate transgenesis by delivering and integrating transgenes into the host genome. Viral vectors can be divided into non-integrating viral vectors e. Serval strategies have been utilized that use of viral vectors as vehicles or carriers to deliver chosen exogenous DNA constructs into targeted expression positions Figure 2.

One of the basic strategies that uses viral vectors is the direct intramammary injection via the teat canal for the transient production of valuable proteins in milk. Goats are an ideal model for the production of pharmaceutical molecules in milk and have been subjected to this protocol.

Human growth hormone hGH was among the first published genes to be infused into the goat mammary gland using retroviral vectors Archer et al. The same strategy has also been applied using adenoviral vectors to direct the expression of functional proteins into mammary secretory epithelial cells. Examples of the adenoviral vector—mediated transfer of genes infused via the teat canal of goat mammary glands are listed in Table 5.

Table 5 Examples of adenoviral-mediated gene transfer into the teat canal of caprine mammary glands. A further strategy that involves the transduction of goat male germline stem cells with an adeno-associated viral vector carrying eGFP marker gene, resulted in transgene transmission after germ cell transplantation Honaramooz et al.

Lentiviral vectors carrying the eGFP marker gene have also been applied to transduce sheep Rodriguez-Sosa et al.

This approach might be a useful tool for the generation of transgenic founders, in particular, since it requires minimal embryo handling. Viral vectors coupled with an RNAi mechanism have been used to mediate the loss of gene expression for the investigation of potential biological functions of genes, to suppress the expression of disease-related genes, and to inhibit the expression of genes that negatively regulate economically important traits.

Lentiviral vector—based delivery of shRNA has also been used in both ovine and caprine cells to suppress the expression of target genes Table 6. Viral vectors can also be used to mediate transgenesis into zygotes by using two main ways: viral transduction of zona-free embryos and perivitelline space subzonal injection Pfeifer and Hofmann, Both strategies have been applied using lentiviral vectors in sheep. Ritchie et al. Perivitelline space injection of lentiviral vectors that carry eGFP marker genes has shown efficient expression of green fluorescence in transgenic lambs Liu et al.

Despite these results, using a 2A peptide—based tricistronic lentiviral vector for the expression of three fluorescent protein genes subjected to hypermethylation and silenced the expression of transgenes in transgenic sheep Tian et al. Furthermore, cells derived from these transgenic sheep could achieve expression of transgenes when the epigenetic status has been regulated via methyltransferase and deacetylase inhibitors Tian et al.

Other interesting examples include the production of transgenic sheep via perivitelline space injection of lentiviral vectors encoding shRNAs that have been specifically designed to inhibit the replication of foot-and-mouth disease virus FMDV , or to silence the expression of myostatin MSTN to promote muscle growth Cornetta et al.

This report also provides additional safety information in support of the application of this technology. Additionally, recombinant lentivirus carrying the fibroblast growth factor 5—short alternative transcript FGF5s was used to produce transgenic sheep with enhanced fiber growth Li et al. Viral vector—based transgenic strategies have shown moderate efficiency compared to other conventional methodologies that have been used to mediate transgenesis.

Despite the moderate efficiency of these strategies, newly emerging gene modifying tools are likely a better choice or are at least simpler and safer to use. Recombinases are enzymes that promote site-specific genetic recombination.

Recombinases are derived from nature and possess the ability to perform deletions, insertions, and inversions into DNA sequences via interaction between the recombinases and their own recognition sites Olorunniji et al. Site-specific recombinases have been integrated into genome engineering programs for a variety of purposes. Manipulations using recombinases have been applied to sheep and goat genomes via Cre recombinase, Flp recombinase, and PhiC31 integrase Figure 2.

This bp sequence consists of two bp inverted or palindromic repeats separated by an 8-bp spacer region Hoess and Abremski, Xu et al. This recombinant protein was used to optimize the efficiency of delivery and to eliminate cytotoxic and genotoxic effects of both the integration and continuous expression of Cre recombinase-expressing vectors Xu et al.

Briefly, Yu et al. Cell clones with successful targeting have been subjected to embryo cloning to achieve rejuvenation or regeneration. Cells with the FRT -homing site were isolated from cloned fetuses and co-transfected with two vectors, an eGFP replacement vector, and an Flp recombinase-expressing vector. After the second round of recombination, transgenic cells exhibited functional expression of eGFP with a gene replacement efficiency of Three pseudo- attP sites were identified in the sheep genome Ni et al.

These pseudo- attP sites were located in intron or intergenic regions. PhiC31 integrase has been applied in sheep Ni et al. Recombinases have been used to perform modifications such as selectable cassette excision or the integration of marker or overexpression cassettes. Further strategies can also be used to achieve desirable modifications in a relatively simple and efficient manner. Transposable elements or transposons have been used for genetic manipulation after highlighting their role as active non-viral DNA delivery systems Largaespada, These systems have been applied to sheep and goat genomes such as the Sleeping Beauty transposon and the PiggyBac transposon Figure 2.

The Sleeping Beauty transposon system works through the mediation of transposase to directly integrate Sleeping Beauty transposon into, mainly, thymine-adenine TA-dinucleotide sites of the target genome Ivics et al. This highlights the potential for the combination of the Sleeping Beauty transposon with RNAi to generate knockdown donor cells for animal cloning.

After pronuclear microinjection, eight out of 92 generated lambs showed positive integration of VP1 -shRNA. Furthermore, Sleeping Beauty transposase and Tn5 transposase were cytoplasmically injected into sheep zygotes to integrate transposon containing recombinant human factor IX rhFIX driven by the BLG promoter Bevacqua et al.

More animals have to be produced for the accurate detection of Tn5 efficiency. Another interesting transposon system, called the PiggyBac , has been applied by our team in cashmere goats. The PiggyBac transposon system was tested to mediate eGFP gene expression and to generate stably transfected cell lines with the use of goat fetal fibroblasts Bai et al. The hair follicle-specific keratin-associated protein 6. Five transgenic cloned founders were produced and showed an increased number of secondary hair follicles that produce the commercially desirable fine cashmere hair.

Transposon systems have shown potential for the mediation of integrations that can result in gene knockdown or gene overexpression.

In addition to the newly emerging toolkits of genetic engineering, transposons can contribute to the generation of desirable genetically modified cell lines and organisms. RNAi is an interesting natural mechanism that has been applied in genetic manipulation programs due to its ability to simply suppress knockdown gene expression by silencing the mRNA of the targeted gene. RNAi has mainly been used to understand the biological and functional roles of specific genes, investigate the inhibitory effects of gene expression, suppress the expression of pathogenic genes, and inhibit the expression of genes that negatively regulate desirable phenotypes.

In sheep and goats, a set of strategies has been used to deliver RNAi-expressing constructs, to mediate the generation of knockdown cell lines, and to obtain transgenic individuals. These methods include the transfection of RNAi-expressing vectors, viral vectors mainly lentiviral vectors , transposons e. To equip sheep and goat models with potential disease resistance, RNAi has been used to suppress the expression of goat prion protein PrP.

The examined brain tissues of a transgenic cloned fetus from lentiviral-shRNA transfected fibroblasts showed a significant decrease in PrP expression Golding et al. These studies highlight the possibilities for the use of RNAi strategies to confer potential disease resistance in farm animals. In addition to providing models with resistance to epidemic diseases, the provision of models with enhanced economical traits is of great importance.

Disrupting the normal function of the negative muscle-mass regulator MSTN offers promising potential for the promotion of meat production. Increasing animal muscle growth and body weight is one of the ultimate goals in agriculture. The MSTN gene is an attractive target to fulfill this purpose. In addition, a set of genes have also been investigated using RNAi in a variety of ovine and caprine cells. Examples of these genes are shown in Table 6.

A further strategy is based on the injection of testis with shRNA vectors, which was performed to target the zinc finger protein Y-linked ZFY gene. This strategy was applied as a genetic method of sex control and to bias the sex ratio toward females in sheep Zhang et al.

In general, RNAi-based approaches are an interesting tool for mediating loss of gene expression by targeting the products of gene transcription mRNA. Currently, more advanced strategies can be easily and directly applied to knockout the gene sequence instead of its mRNA, thus ensuring complete disruption of the associated gene function Figure 3. Figure 3 Gene editing using site-specific endonucleases.