25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER: Current applications of SCNT in advanced breeding and genome editing in livestock

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Cesare GalliAvantea and Fondazione Avantea Onlus, Cremona, Italy

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https://orcid.org/0000-0002-9529-6136
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Giovanna LazzariAvantea and Fondazione Avantea Onlus, Cremona, Italy

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Correspondence should be addressed to C Galli; Email: cesaregalli@avantea.it

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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SCNT (somatic cell nuclear transfer) has complemented the toolbox of ARTs offering yet another technique to reproduce animals in an unprecedented way. Despite remarkable achievements, SCNT suffers low efficiency, high pregnancy losses and higher than normal stillbirth rates that makes it an expensive technique to reproduce animals. Moreover, due to welfare issues associated with gestation and the newborn offspring, it is banned in some countries. It has become evident that these problems are of epigenetic nature associated with incomplete genome reprogramming, observed more frequently in ruminants and less often and of minor degree in pigs and horses. Genome editing is enormously benefiting from SCNT to turn genome edited cells into animals, even if zygote microinjection of CRISPR/Cas9 will become an alternative route in some occasions. SCNT will also be a route to reprogram somatic cell to pluripotency since bona fide iPSC in livestock are missing while embryonic stem cells have been now established. This opens the way to other technologies like the development of artificial gametes or interspecies nuclear transfer. To strengthen its commercial applications, SCNT will face three major challenges, that is, intellectual property (extremely unclear in genome editing), regulatory approval by the relevant authorities of the resuting potential products and finally, acceptance by the public who will eventually decide with its behavior the life or the death of the technology.

Abstract

SCNT (somatic cell nuclear transfer) has complemented the toolbox of ARTs offering yet another technique to reproduce animals in an unprecedented way. Despite remarkable achievements, SCNT suffers low efficiency, high pregnancy losses and higher than normal stillbirth rates that makes it an expensive technique to reproduce animals. Moreover, due to welfare issues associated with gestation and the newborn offspring, it is banned in some countries. It has become evident that these problems are of epigenetic nature associated with incomplete genome reprogramming, observed more frequently in ruminants and less often and of minor degree in pigs and horses. Genome editing is enormously benefiting from SCNT to turn genome edited cells into animals, even if zygote microinjection of CRISPR/Cas9 will become an alternative route in some occasions. SCNT will also be a route to reprogram somatic cell to pluripotency since bona fide iPSC in livestock are missing while embryonic stem cells have been now established. This opens the way to other technologies like the development of artificial gametes or interspecies nuclear transfer. To strengthen its commercial applications, SCNT will face three major challenges, that is, intellectual property (extremely unclear in genome editing), regulatory approval by the relevant authorities of the resuting potential products and finally, acceptance by the public who will eventually decide with its behavior the life or the death of the technology.

Introduction

Advanced animal breeding of livestock in the last decade has undergone major changes with the development and refinements of ARTs (assisted reproduction techniques) enabling the production of more embryos, especially in vitro, in different livestock species (Paramio & Izquierdo 2016, Sirard 2018, Martinez et al. 2019, Lazzari et al. 2020) from selected individuals and the introduction of genomics-based selection in the cattle industry (Ponsart et al. 2013, Jaton et al. 2019). This dramatic change is revolutionizing conventional breeding approaches and the organizations involved, including the artificial insemination industry and the role of individual breeders. The introduction of genomic selection has been made possible by the developments in molecular biology and bioinformatics that allowed better and cheaper genome sequencing and analysis of SNPs (Bickhart et al. 2020). In this new context, ARTs have become the key tools that can utilize the information gained from genomics analysis to increase the number of elite animals with the desired genotype and impact on the following generations in production herds. This is the case for cattle but it is obvious that it will be the case in the future for other livestock species as well. Similarly, the development in more recent years of programmable nucleases for genome editing (Tan et al. 2016) has given new life to the area of genome modifications in livestock that long suffered from the lack of germline competent embryonic stem cells (Navarro et al. 2020). Besides potential agricultural applications for breeding and selection (Jenko et al. 2015, Bhat et al. 2017, Lillico 2019) that still fall short of commercial use, genome editing has even more impacted the biomedical research area: the number of genome-edited livestock lines, primarily pig lines, has grown exponentially for disease models (Luo et al. 2012, Klymiuk et al. 2016), for bioreactors (Bösze et al. 2008, Kuroiwa et al. 2009, Laible et al. 2020) and for xenotransplantation (Perota et al. 2019, Kemter et al. 2020). Twenty-five years since the birth of Dolly, SCNT (somatic cell nuclear transfer) has consolidated its position in the toolbox of advanced assisted reproduction techniques (ARTs) filling a specific niche for particular applications both for breeding and genome editing of livestock. This review will look at the current status of the technology and applications of SCNT in agriculture and medicine and the association with genome editing from a practical and commercial perspective.

The context

SCNT was initially conceived to multiply elite animals for farming purposes, building on the embryo cloning work pioneered by Willadsen (Willadsen 1989). Although the source of cells does not affect pre-implantation embryo developments, the outcome in terms of livebirth is significantly affected. The use of morula stage blastomeres generates embryos after nuclear transfer that have the ability to deliver livebirth at similar rates of embryos obtained by fertilization but this is not the case when fetal or somatic cells are used for the same process (Heyman et al. 2002). The major limitation of embryo cloning is the number of nuclei available from a single pre-implantation embryo. In 1981, Evans and Kaufman reported the establishment in culture of pluripotent cell in the mouse, see Evans (2011) for a review. This discovery fueled the interest in livestock cloning as the availability of an unlimited supply of nuclei for nuclear transfer through embryonic stem cells would overcome a major limitation of the current technology using blastomeres. Unfortunately, the promise of bona fide embryonic stem cells in livestock proved to be an insurmountable task which is only recently being achieved (Bogliotti et al. 2018). Nevertheless, the use of presumptive embryo-derived cells for nuclear transfer had de facto opened the way to SCNT (Campbell et al. 1996). Then after the birth of Dolly (Wilmut et al. 1997), there was a race to clone different species with the more diverse type of somatic cells that reinforced the concept that every cell is suitable as source of nuclei for SCNT. Despite the low efficiency, some of these animals found their way to breeding farms and many were used for research purposes. A few years later, the development of genome editors (programmable nucleases including Zn fingers, TALENS and CRISPR), whose efficacy in editing even somatic cells is exceptional, nicely integrated with SCNT as the major route to generate genome-edited livestock in a research context, with great potential for future developments (Offord 2020) both in the agricultural and biomedical fields.

The technology

Donor cell line

Cell line sourcing is the first key variable in the process of SCNT embryo production and still one of the black boxes responsible for success or failure. Most primary cell cultures from the same type of cells or from different cell types (most frequent are fibroblasts or granulosa cells) have been used with some success in the process. Primary cell cultures have finite lifespan and after a given number of doublings will reach a stage where they undergo aging and stop replication, however, we are far from a full understanding of what are the ideal requirements and the reasons for success or failure in obtaining offspring after SCNT. Culture conditions, doubling numbers, oxygen tension (Mordhorst et al. 2019) etc. can all contribute to the selection during in vitro culture of a particular cell population or a sub-population that influence the status of the chromatin and most importantly its susceptibility to be reprogrammed after nuclear transfer. In general early passages (<6–8) of primary cell cultures are preferred as it is less likely that with increasing population doublings karyotype abnormalities or epigenetic alterations can accumulate over time with extended culture in vitro. Therefore, from a practical point of view, the identification of cell lines with a high SCNT efficiency can lead to astonishing results as opposed to cell lines that deliver huge failures. To gain this information, experiments have to be carried to term with the birth of normal offspring as we do not have other reliable way of selection. All of this work is based on empirical experiments that are time consuming and costly but worth the effort especially if those cell lines are going to be used for genome-editing (GE) projects. The most commonly used cell types are fibroblasts from skin biopsies if the animal to be cloned should be of known genotype/phenotype. If this is not the case, then fetal fibroblasts are the most commonly used cell type especially for GE. There are many reports with claims on the most efficient cell type to be used for cloning pigs (Kurome et al. 2013, Liu et al. 2015) or horses (Olivera et al. 2018) but this choice might be conflicting with the need to use a particular cell line required for a specific project. GE of the cell line to be used for SCNT generally does not change its ability to be used successfully for generating offspring and a slight reduction was observed in the case of gene KO (knock-out) experiments (Kurome et al. 2013, Liu et al. 2015). All cell lines can easily be cryopreserved at early passages before or after GE, ensuring that the same cells can be used over and over again in nuclear transfer rounds while controlling for a key variable in the procedure.

Genome editing

Primary cell lines have a finite lifespan in vitro, undergoing senescence after a certain number of population doublings. This window of time is sufficient to introduce genetic modifications especially now with the use of techniques such as CRISPR/Cas9 (Doudna & Charpentier 2014) that allow to target specific multiple loci at the same time both for inserting (KI, knock-in) or deleting fragments of genes (KO) or to induce homology-directed repair (HDR) with relatively high efficiency compared to random integration or classical homologous recombination. Because this process is carried out on cells cultured in vitro, there are large margins at very limited cost to select amongst many of the cell clone carrying the exact desired mutation. Then, by using SCNT, an animal originating from that genotype can be obtained in a relatively short time, although the direct injection of CRISPR/Cas9 into zygotes can also work in creating GE animals (Lee et al. 2019, Menchaca et al. 2020). But when a multiplexed GE is required, the efficiency of HDR mediated KI is low or the possibility of having a mosaic animal that do not transmit to the offspring (Yen et al. 2014) represent too high risks in a livestock species compared with the direct derivation of an animal by SCNT from the selected cell clone carrying the desired modification.

Embryo production

Over the years, the technique of cloning by nuclear transfer in livestock has not changed in the basic principles pioneered by Willadsen (1986). The first step is the preparation of a matured enucleated oocyte whereby the metaphase plate is removed from matured oocyte by micromanipulation. In the second step, a nucleus coming from an embryonic (embryo blastomere) or somatic cell (primary cell cultures) is transferred by means of micromanipulation in the perivitelline space or stuck to the oolemma with phytohemagglutinin when zona free system is used. Then by means of electrofusion of the two cell membranes, the nucleus is released inside the cytoplasm. Alternatively, the nucleus can be directly injected into the oocyte cytoplasm after breaking the membrane of the donor cell using a piezoelectric micromanipulator. Finally in the third step, electrical or chemical activation is induced to resume the cell cycle in the oocyte. The reconstructed embryos are either transferred at one cell stage to the oviducts (only pig and small ruminants) of synchronized recipients or cultured to the blastocyst stage that can then be cryopreserved or transferred to the uterus. In cattle and horse, the non-surgical transfer is the only practical route, therefore, the embryos need to be cultured to the blastocyst stage in vitro. The large number of metaphase II oocytes required for embryo production in these species can easily be sourced from slaughterhouses at very low cost and in respect of the 3R principle. The procedures to mature oocytes and culture embryos are well established in livestock (Paramio & Izquierdo 2016, Redel et al. 2019, Ferré et al. 2020, Lazzari et al. 2020) and the same are used for SCNT as well. The micromanipulation work is still a bottleneck of the technology as it is labor intensive, requires specialized equipment and above all, experienced embryologists. The metaphase plate can effectively be visualized with Hoechst staining and UV light exposure because the cytoplasm of livestock species is rich in lipids, making them dark compared to the mouse. A scaling up of the nuclear transfer procedure can be to some extent implemented with what is known as handmade cloning (Vajta 2007) or in its various declinations (Oback et al. 2003) by removing the zona pellucida to facilitate enucleation. However, from a practical point of view, being without zona pellucida, it requires that the embryos are cultured in vitro, in special dish to avoid sticking together, to the blastocyst stage prior to transfer. Pre-implantation SCNT embryos do have a reduced potential to develop to term, despite having normal morphology. Developmental competence is depending on another 'black box' that is cellular reprogramming, that is, how the nucleus of the donor cell is reset to direct normal embryo development. At present it is a very inefficient process that has slowly been re-winded essentially in the mouse (Matoba & Zhang 2018) and it is only at the beginning for livestock species to be fully understood (Liu et al. 2018, Ruan et al. 2018). In mouse, significant improvements in livebirth rates have been obtained with the use of Trichostatin A (TSA), a histone deacetylase inhibitor, during the first few hours of culture of the reconstructed embryo after nuclear transfer to help demethylation of the chromatin to favor reprogramming. Similar approaches with a variety of demethylating agents have been explored successfully in some laboratories in the pig (Kishigami et al. 2006) and in bovine where the possible positive effect might be cell line dependent. No data on development to term of bovine embryos were reported (Akagi et al. 2011) and the pig data not always replicated in other laboratories. It is clear that these drugs are not selective for specific targets, therefore, it is possible that imprinted genes can be inevitably affected by the treatment and creating other types of epigenetic problems. Placenta is by far the most-affected organ in most species (Ogura et al. 2013), especially the overgrowth of cotyledonary placentae of ruminants (Chavatte-Palmer et al. 2012) is responsible for reduced rates of implantation and of losses throughout the gestational period including hydrops of the fetal membranes.

Pregnancy

Upon transfer, the ability of SCNT embryos to establish pregnancies is by and large lower than that of embryos obtained by fertilization and this has an economic impact because of the cost of carrying recipients not being pregnant. This can be compensated by the transfer of more embryos as SCNT embryo production usually is not a limiting factor especially in cattle and pigs. In the pig, this is well tolerated, even the transfer of over 100 embryos, since this species can adjust for the number of fetuses developing by physiological reabsorption of the excessive number of embryos developing. Small ruminants can also tolerate twin pregnancy and in the horse, the reduction of twin pregnancies is a routine clinical practice. By contrast, in cattle, twin pregnancies result in premature birth and, therefore, higher stillborn rates. Overall, SCNT pregnancies suffer high losses throughout gestation especially in bovine (Heyman et al. 2002) where they are associated with abnormal placenta development and hydrops (Chavatte-Palmer et al. 2012). When losses occur in advanced gestation, there is a significant animal welfare and economic impact. Another issue with SCNT pregnancies is the prolonged gestation period usually requiring, with the exception of the horse, induction of parturition and again this points to a placental problem.

Offspring

Depending on how the success rate is calculated, on the reconstructed embryos or on the transferred blastocyst, development to term can be up to 13% (Wells et al. 2004), although many variables are responsible of this rate, making comparisons amongst laboratories impossible. Similar or lower success rate is reported for the horse (Galli et al. 2003), the pig (Kurome et al. 2013) and small ruminants (Wilmut et al. 1997, Gavin et al. 2020). In general, SCNT offspring are more fragile animals and perinatal mortality in cattle is twice as much as that of normal calving (Wells et al. 2004). Several pathological features have been identified (Hill et al. 1999) as responsible for the perinatal mortality in cattle but also in the pig, however, in the horse the incidence is rare (Johnson & Hinrichs 2015). To optimize the survival of SCNT-derived offspring, special attention should be given to the late gestation, parturition and neonatal care (IETS 2008, Chavatte-Palmer & Lee 2009). Once the first few days or weeks are over, the cloned animals have a normal life. They are also fertile and, most importantly, the abnormalities observed are not transmitted to their offspring (Cibelli et al. 2002, Tamashiro et al. 2002, Fulka et al. 2004, Heyman et al. 2004). This is an important aspect to be taken into consideration for the commercial application of this technology.

The species and the applications

Bovine/Buffaloes

The main initial purpose of cloning cattle (Bousquet & Blondin 2004) or buffaloes (Selokar et al. 2019) has been to replace elite bulls or dams to have identical copies in case of death, injury, old age of the original animal and/or to meet high market demand of semen or embryos. This is possible in those countries that have approved the use of such products (see below). The use of SCNT-derived animals in agriculture is limited to some countries and no solid data is available in the public domain to have an idea of their impact on the animal population and eventually of their economic return, if any (Faber et al. 2004). Some researchers have been able to follow cloned cows in their production career and reported a 8% mortality rate mainly due to preventable lameness problem; other than that all other parameters were within the normal ranges (Wells et al. 2004). When SCNT is associated with GE, we fall in a research and development area as none of this type of animals have yet or will have, in the short term, a commercial use. GE cattle in the R&D phase are now countless after the initial report (Kuroiwa et al. 2002). Editing for agricultural applications has been limited to single gene character to include mastitis resistance, doble muscling, hornless (polled), β-lactoglobulin, see Bishop and Van Eenennaam (2020) for a review and Wang et al. (2017) on the production of milk being more suitable for some category of the population. Biomedical applications include the use of the animals as bioreactors for producing, for example, human immunoglobulins (Kuroiwa et al. 2009) or as a source of low immunogenic biomaterials (Perota et al. 2019). However, the goat normally replaces the bovine for use as a bioreactor since they are easier to handle/house, they have a shorter generation interval, produce good quantity of milk and a set up for commercial production is more scalable compared to cattle (Wu et al. 2019).

Pig

Although there is a potential interest in the agricultural application of pig cloning (Pratt et al. 2006), there are no published reports of its use by the industry. Disease resistance and double muscling have all been implemented by GE and SCNT, see Bishop and Van Eenennaam (2020) for a review, and also with a modified body composition rich in omega-3 fatty acids (Lai et al. 2006). The pig, however, more than any other large animal has gained its place in the biomedical field initially as potential organ donor for xenotransplantation (Kemter et al. 2020) and now more as an animal model (Aigner et al. 2010) or bioreactor for producing hyperimmune sera (Reynard et al. 2016). Although zygote microinjection of CRISPR/Cas9 is gaining momentum for single gene edit (Lee et al. 2019) because of the high efficiency of gene editor tools, when it comes to multiplexed edits including KO (Niu et al. 2017) and KI (Fischer et al. 2016), the SCNT route with edited cells is still the preferred route.

Sheep

Despite being the first mammal to be cloned, there is no agricultural use of SCNT in sheep, and amongst livestock, it is the species with the lowest efficiency in SCNT (Keefer 2015, Yuan et al. 2019), even if the adult clones are normal (Sinclair et al. 2016). The small value and limited potential impact of any single individual through reproduction does not make SCNT attractive for agriculture use in this species unless novel traits are added through genome editing like double muscling (Zhang et al. 2018). In this species, zygote microinjection seems to be the preferred route to obtain GE animals (Proudfoot et al. 2015, Menchaca et al. 2020). Sheep is also used as disease model (Kalds et al. 2019) but again because of the low efficiency of SCNT and the less-developed ARTs, zygote microinjection appears to be the preferred route.

Goat

The situation in this species is similar to the sheep as the value of cloned individuals for agriculture does not justify the expense of SCNT. Initially the efficiency of cloning goats seemed the highest amongst ruminants (Keefer 2015) in a small dataset, however, now it appears that on larger scale SCNT programs (Gavin et al. 2020) there is not so much difference compared to other ruminants. The agricultural use will make sense if and when GE will be implemented with new traits that will add a relevant economic impact. In the biomedical field, goat has gained acceptance to be used as bioreactor, as goats are good milk producers and easy to handle and production can be scaled up depending on market requirements (Wu et al. 2019, Gavin et al. 2020, Laible et al. 2020). A drug approved by the EMA (European Medicinal Agencies) and FDA (Food and Drug Administration) already exists: Atryn (anticoagulant antithrombin), produced in the milk of goat generated by pronuclear microinjection, was developed before SCNT. This should facilitate the approval of other drugs produced in this species.

Horse

Amongst livestock species, equine is the less problematic to clone although producing pre-implantation embryos is more demanding compared to other species because of the paucity of oocytes available. Because pregnancy losses are usually early during gestation and go unnoticed, they do not cause the welfare issues found in cattle, moreover perinatal losses at birth are very small (Galli et al. 2014). The type and degree of abnormalities seen in bovine are not reported in the horse (Johnson & Hinrichs 2015), especially the placentae are within the normal range. Stallions and brood mares are usually cloned for reproduction purposes. Even more than cattle, individual horses can have extremely high market value justifying the costs involved in cloning them. Gelded males can be very successful in sport but cannot generate offspring, therefore, cloned copies can replace the original in their breeding career as stallions. Injury, disease, death are other possible reasons to clone an individual horse. The value of a horse for reproduction will also depend on its registration in a studbook that can certify its genealogy (Church 2006). Not all breed/studbooks are registering clones despite the fact that in 2012 FEI (Fédération Equestre Internationale) allowed the participation of cloned horses to the Olympic Games, putting its seal on the possibility of using cloned animals for sport and competitions. Polo horses are also cloned and compete in sporting events. Cambiasso, a famous Argentinian polo player, won a prestigious polo match playing the game with six clones of his best mare (Cohen 2016). This is a further proof that cloned animals can be as performing as conventionally bred animals.

The intellectual property (IP)

For any technology to thrive, the intellectual property and the regulatory frameworks for its applications should be in place. Protecting a technology like SCNT is complicated because it is based on principles and techniques developed over many years of research by many investigators. Since there is no full knowledge of how SCNT works, there are many techniques that can be used to achieve the same result and each of them can in theory be the subject of a patent. In addition, IP protection requires a set of different human competences and imposes additional costs both for filing and for maintaining the patent for the 20 years period of its life, including disputes over conflicts with other patents. The first SCNT patent was granted to Geron (Glaser 2000), based on the technology used to create Dolly and was followed by a plethora of patents on cloning technology (Martin-Rendon & Blake 2007). Now that the initial Dolly patent has expired and others, filed later, might soon expire, there should not be restrictions on this side for the commercial use of cloned animals. The situation is more complex if SCNT animals also carry GE traits, especially through CRISPR/Cas9 (Churi & Taylor 2020). It will take years to have a clearer picture of the implications and potential use or constraints of these technologies (Cohen 2020), while the number of patent filings continue to grow (2020).

The regulatory challenges and public acceptance

With or without any IP constrains, for a product of any new technology to be commercialized, regulatory approval is needed as well as the eventual demand from the customers. SCNT is facing all of this despite the fact that cows cloned from blastomeres of a morula (pre-implantation embryo stage) were already registered in the Holstein Association USA and milked together with other cows w/o any issue for many years (Norman et al. 2004). After the birth of Dolly, issues were raised on whether products derived from cloned animals, because of the above-average perinatal mortality of SCNT calves, would be safe to eat. FDA, EFSA (European Food Safety Agency) and other regulatory agencies around the world undertook a long exercise of risk assessment, based on published scientific literature at the time, on meat and milk derived from SCNT cattle, goat and pigs and their offspring (United States Food and Drug Administration 2008, European Food Safety 2012). Both agencies came to the conclusion that products derived from animal clones are not different from those of non-cloned animals. Not only are they not different, but it is also impossible to have a marker that can be used to distinguish them from non-cloned products. Opinions of EFSA and FDA are based on scientific evidence, however, in some geographies such as the European Union (EU), ethics and animal welfare are also taken into consideration as well as public perception assessed by public consultation. As a result, SCNT for agricultural use in the EU is in a limbo: there is no agreement between the EU Commission, who is in favor of approval, and the EU parliament, who instead voted to ban it by a large majority. This stalemate has been dragging on for many years and the proposals to ban it, Proposals 'COM(2013) 892' and 'COM(2013) 893' have finally been withdrawn in September 2020 (2020/C 321/03). Therefore, in the EU, food derived from animal clones currently falls under 'Novel Foods Regulation' (Regulation (EU) 2015/2283) as food derived from animals obtained by non-traditional breeding practices. In this situation, no company in Europe is contemplating bringing products derived from animal clones, or their offspring, to market. In North America, South America and New Zealand, cloning for agricultural purposes is not restricted, however, it is not known if and how much it is used. There is also a consideration that animal clones will be used, given their cost, as breeding stock and only occasionally enter the food chain. See Hur (2017) and van der Berg et al. (2019) for an extensive review on safety aspects and regulations.

The regulatory aspects become even more complex when SCNT is combined with genetic engineering or genome editing. In this case, most countries require specific approval of the GE animal before it is released for commercial use (van der Berg et al. 2020). In the United States and the EU, it will be considered under all circumstances as a GMO and will have to adhere to the relevant regulations. In the United States, it is considered as a new animal drug and has to be approved by FDA. In December 2020, FDA approved the first product from GE animal clones both for human consumption and potential therapeutic use (FDA 2020). Some countries, however, like Brazil, Australia or New Zealand make the distinction if there is a introduction or not of new genetic material. If there is no introduction of recombinant DNA sequences like in the case of KO with CRISPR/Cas9, then they are exempted from GMO regulations. In the case of horse clones that can be registered as non-food animals at birth, the pathway to commercialization might be facilitated, and so could pet animals, as they cannot enter the food chain. The same and even more stringent requirements will apply if the GE is for biomedical applications and the animal-derived products have to enter clinical trials on human patients (Sullivan et al. 2014). Once the necessary regulatory approval to bring the product to market is obtained, its success will depend on public acceptance especially for agricultural applications. However, this is far from granted, especially in the European Union, where consumers have historically been reluctant to accept GMO-based plant products. Biomedical applications, as opposed to introductions to the food supply, are more likely to be accepted.

The future

We have seen in the last 25 years in the life sciences a rapidly evolving field with technologies coming along like SCNT, iPSC (induced pluripotent stem cells), genome sequencing, genome editing, artificial gametes to mention a few, with the promise of changing our lives. With time, we have come to terms with both their potential and their limitations. SCNT is one such example: we know what it can deliver, even if it has not progressed as much as we expected. Nevertheless, it is filling a gap in the ARTs tool box. We know that we can make copies of existing or existed animals, we know that SCNT is an expensive technique because of the low efficiency associated with welfare problems in some species, but it offers unique solutions for niche applications. More research is needed to understand how cell reprogramming works. The oocyte is still the mysterious, fantastic cell that makes it happen and is 'per se' a variable that needs to be taken into account in SCNT (Yuan et al. 2019). Because iPSC have been elusive in livestock (Soto & Ross 2016, Su et al. 2020) and still not up to expectations in humans (Yamanaka 2020), the use of ntESC (stem cells derived from SCNT embryos) (Lazzari et al. 2006) can offer a more reliable route to reprogram somatic cells to pluripotency now that conditions to maintain pluripotency have been established (Hildebrandt et al. 2018, Navarro et al. 2020). Nuclear transfer ESC can then be the route to generate artificial gametes (Hayashi 2019) from selected individuals, to be used in ARTs both in animals – including endangered species – and eventually in humans. Unravelling the mechanisms of cell reprogramming will also solve part of the problems associated with pregnancy losses and stillbirth in SCNT embryos mentioned previously that are the major drawbacks to a wider use of SCNT technology. There are alternative routes to increase the gametes available (only spermatozoa) for breeding purposes based on primordial germ cell culture, with or without GE and transplantation (Giassetti et al. 2019). This approach would require the preparation of a surrogate sire whose germ line is ablated through the knockout of NANOS (a specific gene responsible for donor-derived germ cell development) and then verify that the repopulation with spermatogonial stem cells of the testis results in the production of fertile spermatozoa (Ciccarelli et al. 2020). This is, however, not a viable solution for the female germ line. The alternative route explored to GE by zygote microinjection (Lee et al. 2020, Menchaca et al. 2020) is also not a solution for all projects. When a specific genetic background or a specific breed or genotype already carrying other GE are required, then it is not possible to have sufficient oocytes or zygotes from those females for microinjection and transfer, therefore, SCNT today represents the only workable and proven option. Another area that would be interesting to explore is interspecific SCNT (Lagutina et al. 2013) as this will create opportunities for endangered/extinct species where it is impossible to source oocytes for SCNT and cell reprogramming is a complete failure.

Conclusion

In this review, we outlined the current status of SCNT and the connected genetic modifications that SCNT enables. There are still problems to be solved, in particular, the high pregnancy losses due to reduced viability of the embryos, likely resulting from incomplete reprogramming, especially of the extraembryonic lineages. SCNT is not a solution of all problems but offers important alternative routes in animal reproduction. Besides making copies of animals, it can also reprogram somatic cells into embryonic stem cells that could be used to explore in vitro gametogenesis or to edit the genomes of selected animals. Some applications of SCNT are sufficiently viable to have a market for companies commercializing them as they do not carry an excessive regulatory burden and do not require large investments. Other applications associated with GE editing are still primarily at the R&D phase and will require more time and more investments before they can be commercially viable. In any case, the success on a wider scale of SCNT technology and of the companies offering it will depend not only on solving still-open scientific questions but also on addressing regulatory issues and communicating with the public to increase awareness and trusting in these new technologies.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

Funding

This review was written while funded by INTERSLA project supported by European Union, Italian Government and Lombardy Region POR FESR 2014–2020.

Author contribution statement

C G and G L both conducted the review and wrote the paper.

Acknowledgements

The authors thank Francesca Galli for reading of the manuscript.

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