Abstract
Genetic engineering (GE) of livestock initially has been accomplished primarily using pronuclear microinjection into zygotes (1985–1996). The applications of the technology were limited due to low integration efficiency, aberrant transgene expression resulting from random integration and the presence of genetic mosaicism in transgenic founder animals. Despite enormous efforts to established embryonic stem cells (ESCs) for domestic species, the ESC GE technology does not exist for livestock. Development of somatic cell nuclear transfer (SCNT) has bypassed the need in livestock ESCs and revolutionized the field of livestock transgenesis by offering the first cell-based platform for precise genetic manipulation in farm animals. For nearly two decades since the birth of Dolly (1996–2013), SCNT was the only method used for the generation of knockout and knockin livestock. Arrival of CRISPRS/Cas9 system, a new generation of gene-editing technology, gave us an ability to introduce precise genome modifications easily and efficiently. This technological advancement accelerated production of GE livestock by SCNT and reinstated zygote micromanipulation as an important GE approach. The primary advantage of the SCNT technology is the ability to confirm in vitro that the desired genetic modification is present in the somatic cells prior to animal production. The edited cells could also be tested for potential off-target mutations. Additionally, this method eliminates the risk of genetic mosaicism frequently observed following zygote micromanipulation. Despite its low efficiency, SCNT is a well-established procedure in numerous laboratories around the world and will continue to play an important role in the GE livestock field.
Early cloning experiments
Animal cloning is the process of producing a new organism genetically identical to another organism. The history of cloning is full of exciting experiments and findings. The simplest form of cloning is embryo splitting that can be accomplished by separating the blastomeres of an early embryo and creating two or more smaller embryos. The first artificial embryo twinning was performed by Hans Driesch in 1892. Experimenting with two-cell stage sea urchin embryos, he was able to separate two cells from each other by vigorously shaking a tube of seawater containing embryos. Some of the separated cells grew into complete sea urchins (reviewed in Sander 1992). Hans Spemann, a German experimental embryologist, is often considered the father of cloning. He was studying cellular differentiation in the relatively large eggs of amphibians and made himself a master of microsurgical technique (Spemann 1938, Beetschen & Fischer 2004). In his early study (circa 1902), he reproduced the Driesch’s sea urchin experiment in salamander, showing that embryo splitting results in a normal development in vertebrates. In his later work, he tied a ligature made of baby hair, to restrict the nucleus to a portion of the cytoplasm, creating nucleus-free and nucleus containing fragments. The nucleus containing a portion of the embryo was allowed to divide several times before the ligature was loosened to allow one nucleus to move into the enucleated fragment of the cytoplasm that had not divided. Eventually, the two sections of the embryo each gave rise to normal salamanders. These early cloning experiments affirmed that the nucleus that had divided several times before release into the enucleated fragment had not lost its ability to support growth, at least during that early stage of development (reviewed in Beetschen & Fischer 2004). Spemann could not see how a technical challenge of manually inserting a nucleus from older embryos into enucleated cytoplasm can be accomplished (Spemann 1938), but the stage for modern cloning experiments has been set.
Nuclear transfer, involving the transfer of the nucleus of a donor cell into an oocyte or early embryo from which the genetic material has been removed, had become technically feasible in amphibians in the 1950s. Pioneering studies in frogs by Briggs and King showed that tadpoles developed from some enucleated eggs injected with individual blastula and early gastrula nuclei, and the majority of them metamorphosed into normal juvenile frogs (Briggs & King 1952, King & Briggs 1956). A few years later, John Gurdon demonstrated that nuclei from embryos up to the tadpole stage (somatic cells) were capable of directing normal development, resulting in adult individuals; however, adult cell nuclei in these studies were never able to generate viable adult progeny only tadpoles (Gurdon 1962a,b). Additionally, Gurdon’s experiments demonstrated that the nuclei obtained from earlier developmental stages have a greater ability to be reprogrammed after nuclear transfer into enucleated eggs. For instance, at least 30% of blastula nuclei and only 4% of hatched tadpole nuclei were able to develop to normal adult individuals (Gurdon 1962a). Despite the inability to produce adult frogs after nuclear transfer of adult cells, the studies validated developmental plasticity of differentiated somatic cell nuclei and clearly showed that frog egg cytoplasm contains key factors capable of reprogramming specialized cells to a totipotent state.
Mammalian cloning
Although there was considerable interest in extending cloning to mammalian species, these efforts were delayed by numerous technical challenges, including the development of tools that allow manipulation of small mammalian oocyte (about 100 μm in diameter), the establishment of embryo transfer techniques (Moore & Shelton 1964, Betteridge 1981), and advancements in in vitro culture conditions (Whitten 1956, McLaren & Biggers 1958). The investigators were addressing the same fundamental questions of nuclear potency during embryogenesis that was postulated by researchers earlier for invertebrate and vertebrate species. Additionally, they were seeking to improve livestock species genetics to benefit agriculture.
Similar to the early work in other species, the initial attempts to clone livestock involved embryo splitting (Fig. 1). Steen Willadsen proved that twins could be produced in sheep (Willadsen 1979, 1981) and cattle (Willadsen & Polge 1981) after splitting of cleaved embryos and transfer of the demi-embryos into estrus synchronized recipients. Despite the lower efficiency due to the loss of cytoplasmic volume, triplets and quadruplets were obtained in these studies. To overcome this cytoplasm reduction limitation, a nuclear transfer (NT) technique was developed. Cloning of mammals by NT was initially demonstrated in mice in the early 1980s by transferring either one-cell-stage pronuclei or two-, four-, eight-cell-stage and inner cell mass (ICM) cell nuclei into the cytoplasm of enucleated mouse embryos (McGrath & Solter 1983). Fertilized zygotes were used as recipient cytoplast following the zygote pronuclei removal in these experiments. Later, Robl and First were the first to describe nuclear transfer in pig embryos using a method for pronuclear exchange between zygotes as well as the transfer of nuclei between two-cell stage embryos (Robl & First 1985). Willadsen was the first to report that blastomere nuclei from the 8- to 16-cell stage embryos were able to support full-term development after transfer into enucleated metaphase II (MII) oocytes in sheep and cattle (Willadsen 1986, 1989). Subsequently, several other research groups interested in the agricultural application of cloning achieved success using nuclear donors from morula and ICM of blastocyst stage embryos or using re-cloning procedure (Prather et al. 1987, Stice & Keefer 1993, Collas & Barnes 1994, Keefer et al. 1994, Peura et al. 2001, Keefer 2015).
Timeline of the key milestones in the development of cloning and genetic engineering. The timeline was created using the following research and review papers (Spemann 1938, Briggs & King 1952,Gurdon 1962a, Gordon et al. 1980, Willadsen & Polge 1981, Hammer et al. 1985, Willadsen 1986, 1989, Sander 1992, Campbell et al. 1996b, Schnieke et al. 1997, Wilmut et al. 1997, Cibelli et al. 1998, Baguisi et al. 1999, McCreath et al. 2000, Polejaeva et al. 2000, Dai et al. 2002, Woods et al. 2003, Wani et al. 2010, Tan et al. 2016, Liu et al. 2018, Perisse et al. 2021).
Citation: Reproduction 162, 1; 10.1530/REP-21-0072
The first successful somatic cell nuclear transfer (SCNT) was reported by Keith Campbell et al. in 1996 when he produced two cloned lambs (Megan and Morag) by nuclear transfer using an established differentiated cell line derived from a 9-day-old sheep embryo (Campbell et al. 1996b). One year later, the same technique was applied using adult mammary cells that led to the birth of Dolly, the world’s most famous sheep (Wilmut et al. 1997). This collaborative work between the Roslin Institute and PPL Therapeutics was recognized as the scientific breakthrough of the year (Pennisi 1997) as it broke the existing dogma that specialized somatic cells could not be reprogrammed to the state of totipotency. This groundbreaking research demonstrated for the first time that the genome of a fully differentiated adult cell can be reprogrammed to produce a clone animal. From 1997 to 2000, the SCNT technique was successfully applied for the production of other main livestock species such as cattle (Cibelli et al. 1998), goats (Baguisi et al. 1999) and pigs (Onishi et al. 2000, Polejaeva et al. 2000). One of the primary forces driving the SCNT development was the potential of this technology for the production of genetically engineered livestock.
Technical aspects of nuclear transfer
Techniques for nuclear transfer involve several key steps, each of them potentially has a significant effect on cloning efficiency. These include: (a) removal of metaphase chromosomes from a MII-arrested oocyte (enucleation) by either aspiration or bisection; (b) transfer of donor cell nuclei, in which a donor cell is placed next to an enucleated oocyte and fused by either an electrical pulse or Sendai virus, or a donor cell can be injected directly into the cytoplasm of the enucleated oocyte; (c) activation of the reconstructed embryos; and (d) embryo culture.
Cell cycle coordination during nuclear transfer
Appropriate coordination of cell cycle between the incoming nuclear and recipient cytoplasts is imperative for the nuclear transfer success (Barnes et al. 1987, Collas et al. 1992, Campbell et al. 1993). Since the early attempts of using enucleated bovine zygotes as recipients for blastomere transfer did not result in a competent development (Robl et al. 1987), the MII oocyte has become the primary recipient for NT in livestock. Two distinct methods are available for NT using MII recipient cytoplasts. The first is the transfer of diploid (G1/G0) nuclei into a high MPF (maturation promoting factor) environment of nonactivated MII oocyte. Following the oocyte activation, the donor nuclei will typically experience nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) that take place within 2–4 h after NT. Nuclear envelope is subsequently reconstructed and DNA synthesis is initiated (reviewed in Campbell et al. 1996a, Campbell 1999). S-phase and G2-phase donor nuclei would likely undergo aberrant DNA replication under this high MPF cytoplasmic condition; thus the diploid stage of the cell cycle (G1/G0) must be used to maintain the proper DNA content. The second method is the use of activated MII oocyte with declined MPF activity suitable for any donor nuclei regardless of their cell cycle status ('universal recipient'). The universal recipient approach is preferable for unsynchronized donor cells (e.g. blastomere transfer for which cell cycle synchronization methods are unreliable), while the high MPF MII oocytes are the most desirable recipients for G1/G0 synchronized somatic cells. Notably, the induction of PCC appears to promote nuclear reprogramming (Wakayama et al. 1998, Yin et al. 2003, Choi et al. 2004); however, it is not an absolutely essential requirement for the successful production of cloned offspring (Sung et al. 2007).
Most of the initial SCNT reports were using quiescent somatic cells as nuclear donors. These cells were either induced by serum deprivation to exit replication cycle (Schnieke et al. 1997, Wilmut et al. 1997, Polejaeva & Campbell 2000) or they were in the quiescent (G0) stage of cell cycle in situ (Wakayama et al. 1998). Subsequently, several groups have demonstrated than actively proliferating fibroblasts can be successfully used to generate cloned offspring (Cibelli et al. 1998, Zakhartchenko et al. 1999). However, it is feasible that the animals were obtained from a small fraction of quiescent cells present among the actively dividing fetal fibroblasts (Boquest et al. 1999). In addition, suboptimal culture conditions such as fibroblast clonal expansion and antibiotic selective pressure that are routinely used for the generation of GE fibroblasts for cloning are likely to increase the G0/G1 ratio in the cell population. In a study where quiescent fetal fibroblasts (serum-starved) and actively growing cells from the same cell line were used for SCNT, both blastocyst development rate and pregnancy rate were significantly higher in the serum-starved group, demonstrating a positive effect of G0 on the efficiency of SCNT (Zakhartchenko et al. 1999). Quiescence is characterized by low metabolic activity, active mRNA degradation, transcriptional decline and chromatin condensation (Boynton et al. 1977, Iyer et al. 1999) that might make the chromatin more responsive to reprogramming by oocyte cytoplasmic factors (Campbell 1999). As serum starvation increases a proportion of cells with diploid DNA content and might facilitate cell epigenetic reprogramming, we regularly used this treatment for fibroblast synchronization prior to SCNT (Yang et al. 2016, Fan et al. 2019).
Oocyte activation
Oocyte activation is one of the key technical components of the nuclear transfer procedure. In all species, when MII oocytes are used as recipients, the method of activation is crucial for subsequent development. Under normal condition, the fertilizing spermatozoon induces oocyte activation by generating a transient increase in the intracellular free Ca2+ concentration ([Ca2+]i). Parthenogenic oocyte activation can be induced by a variety of physical and chemical agents (Whittingham & Siracusa 1978, Prather et al. 1999). It can be achieved either by a calcium-dependent mechanism or by a pathway downstream of the calcium signal through inhibition of protein synthesis or kinase inhibition. An increase in ([Ca2+]i can be generated by the entry of external Ca2+ through the oocyte plasma membrane, by exposing the oocytes to electric field pulses resulting in the formation of plasma membrane pores (Zimmermann & Vienken 1982). This method of oocyte activation resulted in the production of viable offspring after the transfer of a nucleus from a four-cell stage embryo (Prather et al. 1989). Another method to increase [Ca2+]i is by stimulating the release of Ca2+ from the smooth endoplasmic reticulum stores through Ca2+ release channels using inisitol 1,4,5-triphosphate (IP3) agonists (Yue et al. 1995). Ca2+, Mg2+ ionophore is able to increase [Ca2+]i. A combination of an increase in [Ca2+]i and inhibition of protein synthesis or protein kinase results in higher rates of pronuclear formation in cattle (Presicce & Yang 1994). This approach is a common activation method for bovine, ovine and caprine oocytes. Species-specific biological differences should be taken into account during SCNT protocol optimization. Oocyte response to artificial activation differs between species depending on the activation type. For instance, porcine oocytes are much more sensitive to electrical stimulation in a Ca2+ Mg2+ containing medium used for fusion compared to bovine and ovine oocytes, and therefore, can be prematurely activated by an electrical pulse intended for fusion. Pre-activation would then lead to an MPF activity decline and reduced development.
Methods of embryo culture, which are not as advanced in pigs, goats and sheep as in cattle, may also play a crucial role in cloning outcomes. To reduce a potentially deleterious effect of suboptimal embryo culture conditions, we limit the culture of reconstructed goat and sheep cloned embryos to 10–12 h. One-cell stage GE cloned embryos are then transferred into estrus synchronized recipients (Fan et al. 2019).
Genetic engineering
Genetic engineering includes a variety of genome modifications such as gene additions (random or site-specific), endogenous gene removal, replacement or disruption, as well as an introduction of specific mutations or single nucleotide replacements.
Random integration using zygote microinjection
In 1980, Jon Gordon demonstrated that genes can be introduced into the mouse genome by direct injection into the zygote pronuclei (Gordon et al. 1980). Using this approach, the first transgenic pigs, rabbits and sheep were produced about 5 years later (Hammer et al. 1985). The strategy of building a transgenic vector involves selecting a genetic regulatory element (promoter) that will determine the tissue in which the gene is to be expressed and the time and level of expression. The second part of the gene construct consists of DNA sequence encoding the desired protein (the structural component). Transgene integration efficiency after pronuclear microinjection is typically lower in livestock (cattle, pigs and sheep) than in laboratory animals (mice, rats and rabbits) averaging 1 and 3%, respectively (reviewed in Wall 1996). The approach had several key restrictions during that time. The inability to delete or modify DNA in situ was the primary constraint (Wilmut & Clark 1991, Pursel & Rexroad 1993). Additionally, a random foreign DNA integration often resulted in an aberrant transgene expression. Furthermore, disruption of endogenous DNA sequences due to the random transgene integration has a potential risk of cellular oncogene activation that could negatively impact the animal's health (Pursel & Rexroad 1993). Finally, mosaicism, when the transgene of interest is not present in all cells (Wieland et al. 1990), is a common feature of GE animals produced by zygote microinjection. Therefore, multiple transgenic founder animals typically need to be produced, followed by establishing transgenic lines by breeding that would eventually allow the validation of germline transmission and completion of phenotype characterization. Although, other methods such as viral vectors and sperm-mediated gene transfer were developed for the production of transgenic mammals, pronuclear microinjection continued to be the primary method of random integration for more than a decade.
Due to poor efficiency, transgenic projects were extremely expensive and thus had limited applications. Production cost for a single transgenic founder was ranging from around $25,000 to over $500,000 for a transgenic pig and a calf, respectively, calculated based on obtaining zygotes from superovulated embryo donors (Wall et al. 1992). The vast majority of transgenic livestock research of the first decade (1985–1996) have focused on the development of human genetic disease models, pharmaceutical protein production and engineering pigs for xenotransplantation. The primary agricultural interest was in livestock growth enhancement and disease resistance (reviewed in Wall 1996).
Targeted integration via homologous recombination
Gene targeting via homologous recombination (HR) using pronuclear microinjection is extremely rare. Brinster et al. obtained only one targeted event out of 10,602 microinjected mouse embryos (Brinster et al. 1989). This low efficiency hampers the practical utilization of pronuclear stage embryos as carriers. Fortunately, the breakthrough discovery of mouse embryonic stem cells (ESCs) (Evans & Kaufman 1981, Martin 1981) offered a powerful platform for genetic manipulations that entirely transformed the field of mouse transgenic research. These pluripotent stem cells have several important characteristics. They are capable of growing indefinitely in vitro while maintaining stable karyotype. Additionally, ESCs have much greater efficiency of HR compare to somatic cells and most importantly, they are able to colonize the germline (Bradley et al. 1984) that opened up unlimited potentials for the precise genetic modification in mice. HR is one of the pathways a cell can use to repair its genome after a double-strand break (DSB). For gene targeting by HR in vitro, a targeting vector is introduced into cultured cells that serves as an alternative template for DSB repair. Both, the first gene targeting in ESCs (Doetschman et al. 1987, Thomas & Capecchi 1987) and the first report of germline transmission from a targeted mutation correction in ESCs (Thompson et al. 1989) were accomplished at the hypoxanthine-guanine phosphoribosyl-transferase gene (HPRT), a selectable locus on the X chromosome. ESC-mediated genetic engineering became the leading technique for gene targeting in mice (Bouabe & Okkenhaug 2013), and currently, there are 7970 phenotyped knockout mouse lines available for researchers (accessed on 29 Jan 2021, https://www.mousephenotype.org/news/data-release-13/) according to the International Mouse Phenotyping Consortium (IMPC), an international organization aiming to identify the function of every protein-coding gene in the mouse genome.
The success in the 1980s with mouse ESC transgenesis stimulated remarkable efforts trying to create livestock ESCs. Numerous research groups reported on the isolation of ESC-like cells for primary livestock species, including cattle, sheep, goat and pig (Notarianni et al. 1991, Galli et al. 1994, Wheeler 1994, Behboodi et al. 2011); however, the initial reports were usually limited to in vitro cell characterization. Limited contribution of porcine putative ESCs to some somatic tissues in chimeras were subsequently documented (Wheeler 1994, Chen et al. 1999, Brevini et al. 2010, Vassiliev et al. 2010). Bovine embryonic stem cells (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time were also recently derived (Bogliotti et al. 2018). Nonetheless, the essential characteristic of mouse ESCs, such as their capability to contribute to germline chimeras (Polejaeva & Mitalipov 2013), has not yet been described for any livestock species.
Cloning enabled the production of first gene-targeted livestock
The birth of Dolly provided the first cell-based methodology for precise genetic manipulation in farm animals; however, gene targeting via HR in primary somatic cells still represented a major challenge. Early homologous recombination experiments were typically performed in transformed human and rodent cell lines (Lin et al. 1985, Smithies et al. 1985, Song et al. 1987). Limited studies have indicated that gene targeting by HR can also be accomplished in primary somatic cells without altering their morphology, karyotype and growth rate (Arbones et al. 1994). However, these reports have been once again restricted to either rodent or human cells. Primary somatic cells have several fundamental differences compared to the mouse ESCs including a limited number of population doublings and low incidence of HR that make gene targeting in somatic cells much more challenging. Somatic cells have about a hundred times lower frequency of HR compared to ESCs (Waldman 1992). To address this inefficiency the targeting vector must offer a powerful enhancement to substantially reduce the presence of non-homologous clones. Several approaches have been developed, including promoterless selection and positive-negative selection. A positive-negative selection (PNS) vector comprises the functionally independent positive and negative selected genes (each with its own promoter and polyadenylation signals) (Sedivy & Dutriaux 1999). The positive selection marker allows picking of all cells with stable vector integration. However, it does not distinguish between homologous or random integration events. Negative selection eliminates the cells containing non-homologous events, thus significantly improving the occurrence of homologous recombination in the resulting colonies (Mansour et al. 1988). Promoterless gene trap vector expressed only if appropriately integrated downstream of an active promoter. Most of the non-homologous insertions are eliminated since the promoterless vector is unable to drive antibiotic resistance. Consequently, the promoter trap method yields targeting efficiency that often exceed 50% (about 100-fold enrichment compared to the traditional gene targeting) (Friedel et al. 2005). The promoterless gene-targeting strategy has been reliably used in primary somatic livestock cells. Although this strategy provides a method to enrich for targeting events, it limits the approach to those genes that are actively transcribed in fibroblasts, the primary cells of choice for SCNT (Denning & Priddle 2003).
A critical requirement for selecting a suitable cell type for HR is that it must be capable of clonal expansion allowing to select for genetically modified clones of interest in vitro. Theoretically, 25 cell doublings are sufficient to produce more than 3 × 107 cells by a single cell clonal growth. Fetal fibroblast cells are commonly used for the production of GE livestock by SCNT as they are easy to establish and maintain in culture and have acceptable longevity in vitro (typically 30–50 population doublings). Nonetheless, most primary somatic cells have an extremely low rate of single cell colony isolation. For instance, individually seeded human dermal fibroblasts are not capable to proliferate under routine culture conditions. Thus, culture condition optimization is essential for successful single-cell colony isolation. Low oxygen culture environment stimulates proliferation and expansion of human adult and neonatal dermal fibroblasts, and also human lung and dermal rodent fibroblasts seeded as single cells (Falanga & Kirsner 1993). Low oxygen cell culture condition (5%O2) is also greatly beneficial for fetal fibroblast single-cell colony isolation in livestock species.
In a short time since the first cloning report, transgenic sheep (Schnieke et al. 1997), cattle (Cibelli et al. 1998) and goats (Keefer et al. 2001) were produced by SCNT (Fig. 1). The approach has also proven to be an effective alternative for ESC technique for the application of HR in farm animals. By the turn of the millennium, SCNT provided a robust method not only for gene addition but also for gene knockout (KO) and gene knockin (KI) in livestock (McCreath et al. 2000, Dai et al. 2002, Lai et al. 2002, Phelps et al. 2003, Kuroiwa et al. 2004) demonstrating that a powerful gene targeting platform has been established for farm animals. However, we were able to accomplish gene KOs and KIs in livestock only by using SCNT during that time. GE livestock research during 1997–2010 has focused primarily on biomedical applications, including xenotransplantation (Klymiuk et al. 2010, Cooper et al. 2014), pharmaceutical protein production and GE animal models of human diseases (Polejaeva et al. 2016a). Agricultural applications have lagged behind primarily due to concerns over the slow regulatory process and public acceptance of livestock containing transgenes in the food chain.
Engineered nucleases
Several types of engineered nucleases have been developed that can generate a DSB at a precise genomic locus: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENS) and clustered regularly interspaced short palindromic repeats (CRISPR). Cell repair machinery is activated to fix the resulting DSBs by either non-homologous end-joining (NHEJ) or homology-directed repair (HDR). NHEJ is the main mechanism of DNA repair. The two ends of the break are brought together and ligated, which often results in the introduction of small insertions/deletions (indels) at the repair site. When the targeted sequence is present in the protein-coding region, indels introduce frameshift of the codon leading to a gene knockout without the requirement for a HR vector. CRISPR/Cas9 system is easy to design for any genomic targets making them extremely versatile. Initially, the NHEJ approach was used to disrupt genes of interest (KO) via indels introduction that was followed by a successful production of KI livestock via HDR using CRISPR/Cas9 base editing, single-stranded oligodeoxynucleotide (ssODN) donor sequences, and CRISPR/Cas9 nickase methods (reviewed by Perisse et al. 2021). The list of newly developed GE tools is growing rapidly. For instance, cytosine and adenine base editors could introduce four types of point mutations and prime editing could introduce all 12 combinations of base changes without creating DSBs (Anzalone et al. 2020). Numerous comprehensive reviews covering the current status of gene-editing in livestock as well as various novel engineered nucleases and their variants are recently published (Tan et al. 2016, Kalds et al. 2020, Navarro-Serna et al. 2020, Perisse et al. 2021). Thus, a summary of several pros and cons of two main procedures used for the production of gene-edited livestock (SCNT and zygote manipulation) is provided here.
Enhancements in CRISPR/Cas9 genome editing over the last several years significantly improved our ability to introduce specific mutations and/or precisely disrupt genes using direct zygote manipulation (most commonly by cytoplasmic injection). A high frequency of indel mutation introduction was recently achieved in mouse, cattle and pig blastocysts following zygotes electroporation (Miao et al. 2019, Navarro-Serna et al. 2020). Zygote electroporation approach eliminates the need in micromanipulation expertise and thus, significantly streamlines production of GE livestock. However, zygote manipulation continues to suffer from accidental genetic mosaicism when the mutation of interest is not present in all cells and/or several types of cells exhibit different mutations (Mehravar et al. 2019). Mosaicism appears when DNA replication takes place prior to gene edition and greatly reduces the likelihoods for direct KO in the resulting offspring. Shortening functional longevity of Cas9 and/or delivering CRISPR/Cas9 earlier into a zygote or MII oocyte could be effective approaches to reduce mosaicism occurrence. In mice, mosaicism was abolished when a Cas9 ribonucleoprotein (RNP) was delivered into early zygotes by electroporation (Kim et al. 2014, Hashimoto et al. 2016). Conversely, mosaicism was not decreased in sheep and cattle following CRISPR/Cas9 injection into MII oocytes compared to the zygote microinjection (Lamas-Toranzo et al. 2019, O'Neil et al. 2020). This indicates that some level of chromatin decondensation might be essential in these species before CRISPR can recognize its target locus. Shortening longevity of Cas9 can be achieved by labeling Cas9 with ubiquitin-proteasomal degradation signals that expedites the nuclease degradation.
The key advantage of the SCNT approach for GE animal production is the ability to verify in vitro that the expected genetic modifications have occurred in the somatic cells that are subsequently used as nuclear donors for SCNT (Fig. 2). Additionally, this method eliminates the risk of mosaicism as the cloned animal is originated from a single GE cell. However, poor development rate (with often only 1–5% of reconstructed embryos developing to viable offspring) limits a broader SCNT utilization. The cloning technique is also considerably more challenging compared to zygote microinjections. Furthermore, cloning related epigenetic errors could be present in the phenotype of GE animals. Thus, the production of F1 generation by breeding is frequently required for an appropriate GE model validation. Despite these SCNT constraints, our latest literature review revealed that SCNT is the main tool for the production of gene-edited livestock via HDR with approximately 70% of reported work used this procedure (Perisse et al. 2021). Also, around 50% of the knockout livestock (NHEJ) were produced by SCNT (Perisse et al. 2021). In my laboratory, we exclusively use SCNT for the production of GE goats and sheep (Fig. 3), and on average, obtain around 45% initial pregnancy rate and 25–35% term development. SCNT results for some of our GE projects are summarized in Table 1. A dozen embryo transfers are usually sufficient to produce a few of GE founder animals for their initial phenotype assessment and GE line expansion by breeding. Intriguingly, we observed significantly greater pregnancy losses and incidence of large offspring syndrome in sheep compared to goats using the same SCNT procedure (Rutigliano et al. 2017). This might be a result of abnormal proinflammatory gene expression in sheep SCNT placentas that were significantly higher than in naturally bred control sheep. Whereas no changes were observed in proinflammatory cytokine gene expression at the maternal-fetal interface in SCNT goats compared to control goats.
Schematic illustration of the production of genetically engineered (GE) farm animals using somatic cell nuclear transfer (SCNT). (A) Somatic cells from the desired donor animal or fetal cell line are cultured and genetically modified in vitro. Then single cell derived colonies are isolated and screened for the presence of genetic modification of interest. (B) Somatic cell nuclear transfer (SCNT): oocytes are recovered from abattoir ovaries and allowed to mature into metaphase (M)II oocytes. MII oocytes are enucleated, and a donor somatic cell is transferred into the perivitelline space of an enucleated oocyte. The cell–cytoplast complexes are fused and activated and then the reconstructed cloned embryos cultured overnight (10–12 h). The embryos are transferred into surrogates and develop into GE cloned offspring. The Figure is assembled using BioRender.com.
Citation: Reproduction 162, 1; 10.1530/REP-21-0072
Genetically engineered goats and sheep produced using genetically modified cells and Somatic Cell Nuclear Transfer. (A) Transgenic goats with cardiac specific overexpression of TGF-β1, a model to study Atrial Fibrillation (Polejaeva et al. 2016b). (B) NANOS2 knockout bucks were subsequently used for spermatogonial stem cell transplantation (Ciccarelli et al. 2020). (C) CFTR+/− sheep used to produce CFTR-null animals by breeding, a model for cystic fibrosis (Fan et al. 2018). (D) A transchromosomic (Tc) goat expressing the human polyclonal antibody, at 2 months of age next to her surrogate mother (Wu et al. 2019).
Citation: Reproduction 162, 1; 10.1530/REP-21-0072
In vivo development of genetically engineered embryos produced by SCNT. We typically transfer 15 ± 4 one-cell stage embryos per recipient, aiming to have two to three viable embryos at the time of implantation. About one-third of the established pregnancies are twin pregnancies and the rest are singletons.
| Species/models | GE type | Embryo transfers, n | Pregnancy rate, n (%) | Term rate, n (%) | GE offspring, n | Reference |
|---|---|---|---|---|---|---|
| TGFβ1 goat model of AF | RI, cardiac expression | 14 | 5/14 (36) | 5/14 (36) | 6 | Polejaeva et al. (2016) |
| CF sheep model (CFTR-/−, CFTR +/−) | NHEJ, KO | 73 | 24/73 (47) | 26/73 (36) | 25 | Fan et al. (2018) |
| Tc goat containing HAC | HAC | 14 | 4/14 (28.6) | 1/14 (7.1) | 1 | Wu et al. (2019) |
| NANOS2−/− goats | NHEJ, KO | 7 | 5/7 (71.4) | 5/7 (71.4) | 5 | Ciccareli et al. (2020) |
| F508delCF sheep model | HDR, KI | 5 | 2/5 (40) | 2/5 (40) | 2 | Perisse et al. (2021)* |
| Total | 113 | 50/113 (44.2) | 39/113 (34.5) | 39 |
*Unpublished observations.
AF, atrial fibrillation; CF, cystic Fibrosis; Tc, transchromasomic; HAC, human artificial chromosome; RI, random integration; GE, genetic engineering; NHEJ, non-homologous end joining; HDR, homology-directed repair.
Further improvements in the efficiency and precision of genetic manipulation and reduction in genetic mosaicism using zygote manipulation would likely expand the utilization of this platform, particularly if zygote electroporation would become highly effective. SCNT is a well-established procedure in numerous laboratories around the world and will continue to play an important role in the GE livestock field due to the benefits outlined above. SCNT is also indispensable for specific GE applications such as an introduction of human artificial chromosome (HAC) to produce transchromosomic animals for human polyclonal antibody production (Wu et al. 2019).
Summary
The initial cloning experiments in both invertebrates and vertebrates used embryo twinning, then later embryonic cells were utilized as nuclear donors to confirm that they are capable to support full-term development. Subsequently, more specialized cells from later stages of development were used as nuclear donors that ultimately led to the birth of Dolly, demonstrating that even fully differentiated adult somatic cells could be reprogrammed by ooplasm to a totipotent status and create an exact copy of the animal they came from. SCNT or cloning has become the method of choice for the production of the transgenic livestock, and by the early 2000s, gene targeting by HR can be accomplished in livestock (Fig. 2).
Remarkable progress has been made over the last two decades in the field of livestock genetic engineering. Initially, knockouts of multiple genes in fetal fibroblasts required in some cases years and were accomplished by sequential targeting and fibroblasts rejuvenation by cloning (Kuroiwa et al. 2004). Arrival of engineered nucleases gave us an ability to introduce precise insertions or deletions easily and efficiently and multiple KOs can be now introduced simultaneously. We also have an ability to change a specific nucleotide in the genome without leaving any additional DNA footprint. Recent advancements in engineered nucleases reestablished zygote micromanipulation as a highly effective GE tool. However, SCNT continues to be the primary method for the production of gene-edited livestock with about 70% of KI and 50% of KO farm animals generated by cloning (Perisse et al. 2021). The primary advantage of genetic engineering via SCNT is the ability to conduct comprehensive cell verification in vitro to ensure that the desired genetic modification is present before GE animal production. The edited cells can also be screened for potential off-target mutations, the presence of which is one of the major concerns (Haeussler 2020). Furthermore, SCNT approach eliminates mosaicism and thus would likely reduce the time requirements and cost of GE animal production. This is particularly important in farm animals that have very long generation intervals (around 5 months in sheep and goats and 9 months in cattle).
The applications of GE livestock biotechnology are extremely versatile, including improving important agricultural production traits, enhancing disease resistance, animal welfare and health. In biomedical field, GE livestock are used for xenotransplantation applications, pharmaceutical protein production and the development of refined animal models of human disease that help us to understand the disease etiology and develop innovative therapeutic procedures (Perisse et al. 2021). Advancements in precision, efficiency and scope of genetic engineering technologies will continue to accelerate GE livestock production and broaden its applications.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Author contribution statement
IAP wrote the manuscript.
Acknowledgements
The author dedicates this review paper to late Professor Keith H.S. Campbell (1954–2012), with who she had the privilege to work closely during the development of pig cloning at PPL Therapeutics Inc. in 1997–2002. This research was supported by the Utah Agricultural Experiment Station (Project 1343) and the USDA/NIFA multistate research project W-4171. The author also would like to acknowledge Iuri Viotti Perisse for helping with illustrative materials. This paper forms part of an anniversary issue on the 25th Anniversary of cloning by somatic cell nuclear transfer. The Guest Editor for this section was Professor Kevin Sinclair, University of Nottingham, UK.
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