25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER Twenty-five years after Dolly: how far have we come?

in Reproduction
Authors:
Bernhard KlingerDepartment of Animal Sciences, Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technical University Munich, Freising, Germany

Search for other papers by Bernhard Klinger in
Current site
Google Scholar
PubMed
Close
and
Angelika SchniekeDepartment of Animal Sciences, Chair of Livestock Biotechnology, School of Life Sciences Weihenstephan, Technical University Munich, Freising, Germany

Search for other papers by Angelika Schnieke in
Current site
Google Scholar
PubMed
Close

Correspondence should be addressed to A Schnieke; Email: angelika.schnieke@wzw.tum.de

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 Prof Kevin Sinclair, University of Nottingham, UK

Free access

For more than a century, the scientific consensus stated that a nucleus from a terminally differentiated cell would not be able to control the development of offspring. This theory was refuted by the birth of Dolly, the first animal generated by nuclear transfer using an adult somatic cell as a nuclear donor. Following this paradigm shift, a wide variety of animals has been cloned using somatic cell nuclear transfer. Coupled with modern genome engineering technology, somatic cell nuclear transfer has become the method of choice for the generation of genetically modified farm animals. This has opened new opportunities to study the function of genes and has led to the establishment of animal models for a variety of human conditions and diseases or to improve the health of livestock animals.

Abstract

For more than a century, the scientific consensus stated that a nucleus from a terminally differentiated cell would not be able to control the development of offspring. This theory was refuted by the birth of Dolly, the first animal generated by nuclear transfer using an adult somatic cell as a nuclear donor. Following this paradigm shift, a wide variety of animals has been cloned using somatic cell nuclear transfer. Coupled with modern genome engineering technology, somatic cell nuclear transfer has become the method of choice for the generation of genetically modified farm animals. This has opened new opportunities to study the function of genes and has led to the establishment of animal models for a variety of human conditions and diseases or to improve the health of livestock animals.

Introduction

The concept of somatic cell nuclear transfer (SCNT) was originally introduced in 1938 as a means to study cell differentiation (Spemann 1938). Pioneering work by Briggs and King (1952) conducted in amphibians led to the generation of the first cloned animals from cells at the blastocyst stage (Briggs & King 1952) but failed when using cells from the late gastrulation stage (King & Briggs 1955). That led to the conclusion that irreversible chromosomal changes had occurred and nuclear totipotency had been lost. In 1962 Gurdon conducted the first successful SCNT – epithelial cells from tadpoles – to generate living tadpoles (Gurdon 1962). It took 21 more years until the first nuclear transfer was performed in mice (McGrath & Solter 1983). The donor cell was derived from a single cell embryo, the recipient cell was a zygote. In contrast, blastomeres from four-cell or later stage embryos did not support embryo development. Again, it was stated that 'the inability of cell nuclei from these stages to support development reflects rapid loss of totipotency'. A couple of years later, the birth of the first sheep cloned from an eight-cell stage blastomere was reported using unfertilised oocytes instead of zygotes as recipient cells (Willadsen 1986). Other mammalian species such as rabbits (Stice & Robl 1988), pigs (Prather et al. 1989), and cattle (Prather et al. 1987) followed shortly thereafter. However, all of these animals were cloned using blastomeres from early embryos as nuclear donors. In 1996 the birth of sheep derived from cultured inner cell mass cells was reported (Campbell et al. 1996). In the same year Dolly, the first cloned offspring generated from an adult-derived somatic cell population, was born (Wilmut et al. 1997). The birth of Dolly irrevocably dispelled the longstanding hypothesis that cell differentiation involves irreversible modifications that make cell dedifferentiation and therefore cloning of mammals impossible (Weismann et al. 1893).

Despite its success, technical problems have made the practical application of somatic cell nuclear transfer (SCNT) technology challenging. First, Dolly was the only offspring obtained from 277 reconstructed embryos (efficiency of 0.3%) (Wilmut et al. 1997). Since then, the efficiency of the cloning procedure has improved for some species (e.g. cattle) but remained low (<5%) for many other species (e.g. sheep, mouse, pig, rabbit) (Meissner & Jaenisch 2006). Second, malformations are frequently observed in cloned embryos and their extraembryonic tissues (Ogura et al. 2013). Both are caused by faulty or insufficient epigenetic reprogramming. Mechanisms involved in reprogramming have been reviewed by Matoba et al. (Matoba & Zhang 2018) and insights gained have led to approaches on how to overcome these defects/problems (Cibelli & Gurdon 2018).

SCNT is first of all a reproductive technology that enables the preservation and rederivation of elite animals (Galli et al. 2003) or endangered species (Lanza et al. 2000). However, its greatest potential has been aa simple fact that cells could be cultured, expanded, and precisely modified prior to nuclear transfer. It opened novel avenues for livestock breeding, fast-track introduction of known (hornless cattle (Carlson et al. 2016)), or novel traits such as disease resistance for example, porcine reproductive and respiratory syndrome virus (PRRSV) (Prather et al. 2017). By facilitating the generation of genetically modified animals, SCNT also holds great potential for biomedical research (Rogers 2016). Here the pig is especially well suited due to its similar size, anatomy, and physiology to humans (Perleberg et al. 2018).

In this review, we give a brief historical perspective on what has been achieved in the field of SCNT in the last 25 years. We discuss its advantages and disadvantages as a tool for the generation of genetically modified animals, provide examples of its application, and ask if novel technologies have superseded SCNT.

Cloning technology 25 years of incremental advances

SCNT is a multi-step process that comprises the generation of a cytoplast via enucleation of an oocyte, selection of a suitable donor cell, embryo reconstruction, activation, and culture. Mature oocytes in metaphase II of the second meiotic division (MII) are the cytoplast donor of choice as their use results in the highest developmental potential. In the past MII oocytes for the production of cytoplasts had to be sourced from donor animals but for many species, they can now be replaced by in vitro matured oocytes. This is especially relevant for domestic animals where large numbers of oocytes can be obtained from slaughterhouse material. The quality of in vivo derived cytoplasts is higher but commonly involves superovulation regimes to increase yield, which adversely impacts embryo development and foetal growth (Van der Auwera I & D'Hooghe 2001). In accordance with the 3R principle, the substitution of in vivo with in vitro matured oocytes significantly reduces the number of experimental animals required (Yuan & Krisher 2012). If the goal is the preservation of endangered species with an inevitable lack of oocytes, those from closely related species can serve as suitable cytoplast donors (Lanza et al. 2000). A timeline depicting the development of cloning related technology is shown in Fig. 1.

Figure 1
Figure 1

The development of SCNT and related technology.

Citation: Reproduction 162, 1; 10.1530/REP-20-0652

Enucleation

Cytoplasts are generated in a process termed enucleation, in which the genetic material is removed from the recipient cell. Enucleation can be performed blindly by removing the first polar body and about one-third of the cytoplasm beneath it with a glass pipette. However, in many cases the metaphase plate is not located close to the polar body (Nour & Takahashi 1999). Furthermore, the removal of a significant proportion of the cytoplasm reduces the oocytes' capacity for epigenetic reprograming and subsequent embryonic development. Successful enucleation can be confirmed by exposing recipient oocytes to UV light after staining with DNA specific fluorochromes but this approach decreases the viability of the resulting cytoplast (Dominko et al. 2000). Enucleation can also be performed chemically, for example with ethanol and demecolcine, but this reduces embryonic development compared to mechanical enucleation methods (Gasparrini et al. 2003).

Embryo reconstruction, activation and culture

Embryo reconstruction by fusion of donor and recipient cells using a DC electric pulse is the method of choice in most species. Piezo- or laser-assisted microinjection is the most common technique in rodents because electrofusion was less successful in those species due to the smaller size and fragility of the oocytes (Wakayama et al. 1998). The reconstructed embryo then has to be activated to exit meiotic arrest and initiate embryonic development. Chemical activation can be achieved by exposure to strontium in mice and ionomycin in cattle or sheep (Akagi et al. 2003). Electrical activation remains the most widely used method in livestock (Polejaeva et al. 2000). The exact activation stimuli, timings, and post-activation treatments must be optimised for each individual protocol and species.

Embryo culture conditions and the stage at which reconstructed embryos are transferred to recipients are species-specific. In vitro cultivation of embryos increases the incidence of epigenetic defects, which is typically associated with developmental abnormalities such as large offspring syndrome (LOS) (Young et al. 1998). LOS is characterised by overlarge foetuses, placental malformations, reduced pregnancy rates, dystocia, and pulmonary dysfunction (Fleming et al. 2004). Pathologies based on an altered epigenome are largely contributed to suboptimal culture conditions, such as medium containing serum (Hill 2014). The adverse consequences of in vitro culture can be minimised by transferring reconstructed embryos at an early stage of development to the oviduct of the recipient. This approach is practicable in mice and pigs (Wakayama et al. 1998, Polejaeva et al. 2000). However, for most domestic animals, this is not an option because small litter sizes, high costs, and long gestation intervals make the availability of recipients the limiting factor. Therefore, reconstructed embryos must be cultured to the blastocyst stage in vitro to ensure that only viable embryos will be transferred.

Handmade cloning

Handmade cloning (HMC) is an alternative cloning technique that forgoes the use of micromanipulators. The characteristic feature of this method is that enucleation of oocytes is conducted with a handheld blade after partial zona pellucida digestion (Vajta et al. 2003). The site of the metaphase plate is indicated by the polar body or can be determined by demecolcine treatment which leads to the formation of a protrusion cone (Vajta et al. 2003). Bisection is conducted manually with an embryo-splitting blade to generate a cytoplast and a karyoplast. Another distinguishing aspect of HMC is that two cytoplasts are fused with the donor cell instead of one. This counteracts the typical loss of cytoplasm during enucleation, which has a positive effect on embryo development (Sayaka et al. 2008). Zona-free embryos require special culture conditions in a well of the well system before they can be transferred to recipients at the blastocyst stage. With this approach, mitochondria from three different animals are introduced into one individual but so far, no deleterious effects of this heteroplasmy have been reported (Steinborn et al. 2000). HMC has the advantage that only minimal equipment is required, reducing the cost of cloning while providing similar cloning efficiency (Tecirlioglu et al. 2005).

A method for all mammals

Other than the development of zona-free approaches mentioned above, cloning technology has remained largely the same since it was first described in 1986 (Willadsen 1986). The greatest advance has been the adaptation of SCNT technology to other species. For decades embryonic stem cells (ESCs) had been the only cell-based method for the generation of animals. Shortly after the publication of Dolly, SCNT technology expanded first to other domestic species, followed by wild cats, wolves, and other endangered species (Gomez et al. 2004, Kim et al. 2007). Certain species are notoriously hard to clone. Dogs, for example, have a very limited breeding period, in vitro maturation is difficult and their oocytes are coated with fat which impedes enucleation (Lee et al. 2005). In pigs at least four foetuses are required to maintain a pregnancy (Prather 2000). Consequently, most technological advances have been species-specific. The main obstacle for some species remains the lack of sufficient numbers of oocytes. To date, a large variety of mammalian species including monkeys (Liu et al. 2018) have been cloned by SCNT using a wide range of donor cell types from foetal and adult tissues (see Fig. 2).

Figure 2
Figure 2

Timeline of cloned mammalian species over the last 25 years.

Citation: Reproduction 162, 1; 10.1530/REP-20-0652

Beyond reproductive cloning

In mice pluripotent ESC based chimera technology offers an elegant way for the generation of Genetically modified (GM) animals (Capecchi 1989b). ESCs are genetically modified in cell culture and injected into a recipient blastocyst followed by transfer to a pseudo-pregnant female to gestate chimeric offspring (Capecchi 1989b). Appropriate breeding results in mice carrying the desired genotype (Koller & Smithies 1992). ESCs are a powerful tool for gene targeting because they maintain pluripotency and a normal karyotype during long term culture and support homologous recombination (HR) at a high frequency (Capecchi 1989a). Pluripotent ESCs with proven germline transmission are currently still unavailable in livestock even though there are promising reports for sheep, cattle, and pigs (Bogliotti et al. 2018). It has been the lack of ESCs which spurred research into SCNT as an alternative method for the generation of GM offspring in domestic species.

Genetically modified (GM) animals hold great potential for agriculture and biomedical research. Mice are the most widely used laboratory animals because they are inexpensive to house and techniques for their genetic modification are well established. However, rodents often do not mimic human disease pathology or phenotypes accurately which makes large animal models indispensable (Mak et al. 2014). Similarities in body size, organ anatomy, diet, and pathophysiology make the pig the animal of choice to model human diseases. Pigs are highly fertile, housing conditions including specific-pathogen-free (SPF) are well established and public acceptance for the use of livestock in animal experiments is less controversial than for primate species or companion animals. Porcine disease models have been generated for cancer research (Flisikowska et al. 2013), diabetes (Wolf et al. 2014), cystic fibrosis (Rogers et al. 2008), and Duchene muscular dystrophy (Klymiuk et al. 2013). Pigs with multiple genetic modifications may in the near future serve as donors for xeno-organ transplantation into humans (Fischer et al. 2016). Furthermore, Cattle, sheep, and goats have been proposed as bioreactors for the production of pharmaceutically important proteins (Schnieke et al. 1997). Genome engineering also has the potential to revolutionise animal breeding (Ruan et al. 2017), improve productivity, animal welfare, reduce the use of antibiotics, and protect the environment (Laible et al. 2015).

Genetic engineering in livestock

Pronuclear DNA microinjection was the first method for the generation of GM large animals but this approach is inefficient, facilitates only random integration of transgenes, and frequently leads to mosaicism (Hammer et al. 1985). Viral vectors provided a more efficient alternative, but they only allow for limited transgene size and the preparation and concentration of viral particles is time and labour-intensive (Reichenbach et al. 2010). Transposon-mediated transgenesis is efficient and has advantages over viral vectors regarding cargo size, ease of implementation, and biosafety. However, this approach depends on microinjection as a delivery method and again only allows for random integration of transgenes (Zhao et al. 2016). Sperm-mediated gene transfer (SMGT) employs the natural ability of sperm to introduce DNA into oocytes to co-transfer exogenous DNA. Theoretically, this makes micromanipulation, culture, and transfer of embryos obsolete but despite its simplicity, successful implementation of SMGT has been limited to few laboratories worldwide, rendering its validity questionable (Brinster et al. 1989).

In contrast to the methods mentioned above, SCNT enabled both the generation of transgenic animals (Schnieke et al. 1997) and, more importantly, gene-targeting (McCreath et al. 2000). The method, first established in sheep, was soon extended to other species (Polejaeva et al. 2000) and remained the most frequently used tool for the generation of GM domestic animals. Extensive selection and screening of donor cells ensure that 100% of offspring carry solely and precisely the intended modification (Wolf et al. 2000). This is a considerable advantage over ESC technology because the injection of ESCs into blastocysts results in chimeric offspring. Consequently, germline transmission has to be assessed by breeding which is not realistic in species with single offspring and long gestation times.

A major disadvantage of SCNT has been the fact that gene targeting in somatic cells has been orders of magnitude less efficient than in pluripotent stem cells. This changed with the emergence of nuclease-based genome editing technology, which has further expanded the toolbox for the generation of genetically modified animals. Site-specific endonucleases, such as zinc finger nucleases, transcription activator-like effector nucleases, and the CRISPR/Cas9 system facilitate the introduction of double strand breaks (DSBs) into predetermined sites within the host genome to trigger DNA repair mechanisms, thus enabling efficient genome engineering (Gaj et al. 2013). The power of this technology has been demonstrated by the inactivation of multiple endogenous copies of the same retroviral gene (Niu et al. 2017) or simultaneous inactivation of several different host genes (Fischer et al. 2019) in somatic cells, followed by the generation of cloned animals. Due to their high-efficiency genome editing reagents, for example, CRISPR/Cas9 can also be delivered directly to early embryos by intracytoplasmic microinjection (Carbery et al. 2010) or electroporation (Hashimoto & Takemoto 2015). Genome editing in zygotes is a powerful method for the inactivation of genes but had been less efficient at introducing modifications via homology directed repair (HDR). New strategies are being developed to improve efficiencies (Nambiar et al. 2019). One limitation is the high incidence of mosaicism which is especially problematic in livestock due to the long generation interval (Ryu et al. 2018). Another downside of Cas9 and other site-specific nucleases are possible off-target effects, that is, the induction of DSBs at unwanted locations (Anderson et al. 2018). Genome editing in combination with SCNT allows for comprehensive in vitro off-target analysis prior to the generation of animals, avoiding undesirable mutations as well as mosaicism (Zhang et al. 2015). The high adaptability, usability, and efficiency of modern genome engineering technology has revolutionised the production of GM animals (Sander & Joung 2014). The individual strengths and weaknesses of both approaches complement each other well, and together, they provide an efficient toolkit for the generation of GM livestock (see Fig. 3).

Figure 3
Figure 3

The toolkit for the generation of genetically modified livestock. (A) Pronuclear DNA microinjection leads to random transgene integration but does not facilitate gene targeting. Viral vectors and transposons can be used to increase the frequency of transgenesis. (B) Cytoplasmic microinjection of site-specific endonucleases facilitates targeted genome engineering directly in early embryos. (C) Genetically engineered somatic cells selected for the presence of the desired modification. Single cells are inserted into the perivitelline space of enucleated oocytes, followed by cell fusion and activation to generate reconstructed embryos for embryo transfer.

Citation: Reproduction 162, 1; 10.1530/REP-20-0652

SCNT – has it been superseded?

SCNT is a reproduction method. In the absence of ES cells, it has been the only method to re-derive animals from cultured cells. After more than 30 years of effort, one can be cautiously optimistic that fully functional ES cells might soon be available for all livestock species. However, except for the pig, the generation of chimeric animals is not a realistic option for most large animal species. The alternative could be tetraploid aggregation to obtain non-chimeric offspring, which is the gold standard to assess ES cell functionality (Nagy et al. 1990). Time will tell if this can be achieved for livestock ES cells. Until then, livestock ES cells or their differentiated derivatives might serve as donor cells for SCNT. In contrast to somatic cells, ES cells have the advantage that they grow permanently in culture and allow for efficient targeted modification.

When it comes to precise genome engineering, genome editing has become the method of choice. It is developing at a fast-pace and has replaced SCNT for some applications. More often, for complex genome modifications as required for xeno-organ transplantation, it is a combination of tools, including SCNT and CRISPR/Cas9, that is required.

A recent publication suggested donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout male pigs, goats, and cattle as a tool for biomedical research, preservation of endangered species, and dissemination of desirable genetics (Ciccarelli et al. 2020). While this approach is limited to the preservation of male genetic material, it could provide an alternative to reproductive cloning once efficiencies of donor-derived spermatogenesis have been improved (Ciccarelli et al. 2020). In the future, it may also allow for the generation of GE livestock. Culture conditions for spermatogonial stem cells exist for some species and after transduction with viral vectors, and transplantation into the testis murine spermatogonial stem cells supported the development of transgenic offspring (Kubota et al. 2004).

In 2008 the Wakayama’s group reported the resurrection of a dead mouse, frozen at −20oC for 16 years by establishing an ESC line from cloned embryos using brain nuclei as nuclear donors followed by a second nuclear transfer using the ES cells (Wakayama et al. 2008). The result was somewhat surprising as brain cells are generally poor donor cells; on the other hand, the high levels of endogenous brain glucose may have acted as a cryoprotectant. This amazing achievement brought the notion that extinct species might also be brought back to life by SCNT, possibly from samples conserved in permafrost (e.g. mammoth) or using a combination of genetic engineering and SCNT (Sherkow & Greely 2013). Resurrection biology made the headlines but due to technical, environmental, and especially ethical considerations, it may never be realised for prehistoric mammals. However, for some species at the brink of extinction SCNT is a reproductive option (Folch et al. 2009).

Soon after the birth of Dolly, SCNT was suggested for the generation of patient-specific ES cells (ntESCs) (Tachibana et al. 2013). At the time, it was the only method to obtain autologous pluripotent stem cells for regenerative medicine or the correction of congenital defects. Besides technical and ethical hurdles, the shortage of human oocytes was the main obstacle. In 2006 Yamanaka showed that a transient expression of four transcription factors (Oct4, Sox2, Myc, Klf4) is sufficient to dedifferentiate cells to an induced pluripotent stem (iPS) cell state (Takahashi & Yamanaka 2006). IPS cells are functionally equivalent to ES cells and can be isolated from patients’ cells without any of the drawbacks of ntESCs which makes therapeutic cloning obsolete. However, cloning technology is still relevant for the treatment of human inherited mitochondrial diseases (Zhang et al. 2017). Mitochondria are the powerhouse of the cell, mitochondrial DNA (mtDNA) is maternally inherited and dysfunction due to mutations (e.g. Leigh’s syndrome) can therefore be detrimental to foetal development. It is avoidable if the mother's genome is transferred into an enucleated healthy recipient oocyte (spindle transfer) followed by fertilisation.

Concluding remarks

The birth of Dolly 25 years ago proved to the world that somatic cell differentiation is reversible. It was exciting science and on a personal note taught me the importance of open discussions of science in progress. Hearing from Jim McWhir over a cup of tea about the experiment that later resulted in the birth of the cloned sheep Megan and Morag (Campbell et al. 1996) got me involved in the project as a Ph.D. student. Talking to Alex Kind about expressing milk proteins in cell culture led to the use of mammary cells as the nuclear donor that gave rise to Dolly. Those more experienced than myself were sceptical because it was a high-risk project. Luckily it succeeded – with a sample size of one – thanks to the work of Ian Wilmut’s and Keith Campbell’s group. It couldn’t have been realised without financial support which was granted by Alan Colman who was at the time scientific director of PPL-Therapeutics. If I – a youngish unknown scientist – would have had to apply for a public grant to disprove a dogma, would it have been approved? I doubt it. Dolly was the result of teamwork. All involved, including all technical personnel, played an essential part. Sadly, two people, Keath and Jim, are no longer with us.

A quarter of a century ago, when the Dolly paper was published, my young son was baffled – why all the excitement about one more sheep when there were so many sheep in Scotland? It was a very special sheep, it had a name, and Dolly caught the imagination not only of the scientific world but also the public. It spurred an interest in science. It taught us that all cells can be reprogrammed, which ultimately led to the development of iPS cells. Until recently cloning has been the only option to precisely alter the genome of many species. Has it become an obsolete technology with the rise of CRISPR/Cas9 or livestock ES cells? Possibly at some point in the future but not yet. If you are visiting Scotland, go and see Dolly in the Royal Museum in Edinburgh.

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.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Author contribution statement

B K and A S wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

References

  • Akagi S, Adachi N, Matsukawa K, Kubo M & Takahashi S 2003 Developmental potential of bovine nuclear transfer embryos and postnatal survival rate of cloned calves produced by two different timings of fusion and activation. Molecular Reproduction and Development 66 264272. (https://doi.org/10.1002/mrd.10352)

    • Search Google Scholar
    • Export Citation
  • Anderson KR, Haeussler M, Watanabe C, Janakiraman V, Lund J, Modrusan Z, Stinson J, Bei Q, Buechler A & Yu C 2018 CRISPR off-target analysis in genetically engineered rats and mice. Nature Methods 15 512514. (https://doi.org/10.1038/s41592-018-0011-5)

    • Search Google Scholar
    • Export Citation
  • Bogliotti YS, Wu J, Vilarino M, Okamura D, Soto DA, Zhong C, Sakurai M, Sampaio RV, Suzuki K & Izpisua Belmonte JC 2018 Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. PNAS 115 20902095. (https://doi.org/10.1073/pnas.1716161115)

    • Search Google Scholar
    • Export Citation
  • Briggs R & King TJ 1952 Transplantation of living nuclei from blastula cells into enucleated frogs' eggs. PNAS 38 455463. (https://doi.org/10.1073/pnas.38.5.455)

    • Search Google Scholar
    • Export Citation
  • Brinster RL, Sandgren EP, Behringer RR & Palmiter RD 1989 No simple solution for making transgenic mice. Cell 59 239241. (https://doi.org/10.1016/0092-8674(8990282-1)

    • Search Google Scholar
    • Export Citation
  • Campbell KH, Mcwhir J, Ritchie WA & Wilmut I 1996 Sheep cloned by nuclear transfer from a cultured cell line. Nature 380 6466. (https://doi.org/10.1038/380064a0)

    • Search Google Scholar
    • Export Citation
  • Capecchi MR 1989 aAltering the genome by homologous recombination. Science 244 12881292. (https://doi.org/10.1126/science.2660260)

  • Capecchi MR 1989 bThe new mouse genetics: altering the genome by gene targeting. Trends in Genetics 5 7076. (https://doi.org/10.1016/0168-9525(8990029-2)

    • Search Google Scholar
    • Export Citation
  • Carbery ID, Ji D, Harrington A, Brown V, Weinstein EJ, Liaw L & Cui X 2010 Targeted genome modification in mice using zinc-finger nucleases. Genetics 186 451459. (https://doi.org/10.1534/genetics.110.117002)

    • Search Google Scholar
    • Export Citation
  • Carlson DF, Lancto CA, Zang B, Kim ES, Walton M, Oldeschulte D, Seabury C, Sonstegard TS & Fahrenkrug SC 2016 Production of hornless dairy cattle from genome-edited cell lines. Nature Biotechnology 34 479481. (https://doi.org/10.1038/nbt.3560)

    • Search Google Scholar
    • Export Citation
  • Cibelli JB & Gurdon JB 2018 Custom-made oocytes to clone non-human Primates. Cell 172 647649. (https://doi.org/10.1016/j.cell.2018.01.030)

  • Ciccarelli M, Giassetti MI, Miao D, Oatley MJ, Robbins C, Lopez-Biladeau B, Waqas MS, Tibary A, Whitelaw B & Lillico S 2020 Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males. PNAS 117 2419524204. (https://doi.org/10.1073/pnas.2010102117)

    • Search Google Scholar
    • Export Citation
  • Dominko T, Chan A, Simerly C, Luetjens CM, Hewitson L, Martinovich C & Schatten G 2000 Dynamic imaging of the metaphase II spindle and maternal chromosomesin bovine oocytes: implications for enucleation efficiency verification, avoidanceof parthenogenesis, and successful embryogenesis. Biology of Reproduction 62 150154. (https://doi.org/10.1095/biolreprod62.1.150)

    • Search Google Scholar
    • Export Citation
  • Fischer K, Kraner-Scheiber S, Petersen B, Rieblinger B, Buermann A, Flisikowska T, Flisikowski K, Christan S, Edlinger M & Baars W 2016 Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing. Scientific Reports 6 29081. (https://doi.org/10.1038/srep29081)

    • Search Google Scholar
    • Export Citation
  • Fischer K, Rieblinger B, Hein R, Sfriso R, Zuber J, Fischer A, Klinger B, Liang W, Flisikowski K & Kurome M 2019 Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2. Xenotransplantation 27 e12560. (https://doi.org/10.1111/xen.12560)

    • Search Google Scholar
    • Export Citation
  • Fleming TP, Kwong WY, Porter R, Ursell E, Fesenko I, Wilkins A, Miller DJ, Watkins AJ & Eckert JJ 2004 The embryo and its future. Biology of Reproduction 71 10461054. (https://doi.org/10.1095/biolreprod.104.030957)

    • Search Google Scholar
    • Export Citation
  • Flisikowska T, Kind A & Schnieke A 2013 The new pig on the block: modelling cancer in pigs. Transgenic Research 22 673680. (https://doi.org/10.1007/s11248-013-9720-9)

    • Search Google Scholar
    • Export Citation
  • Folch J, Cocero MJ, Chesne P, Alabart JL, Dominguez V, Cognie Y, Roche A, Fernandez-Arias A, Marti JI & Sanchez P 2009 First birth of an animal from an extinct subspecies (Capra pyrenaica pyrenaica) by cloning. Theriogenology 71 10261034. (https://doi.org/10.1016/j.theriogenology.2008.11.005)

    • Search Google Scholar
    • Export Citation
  • Gaj T, Gersbach CA & Barbas CF 3RD 2013 ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology 31 397405. (https://doi.org/10.1016/j.tibtech.2013.04.004)

    • Search Google Scholar
    • Export Citation
  • Galli C, Lagutina I, Crotti G, Colleoni S, Turini P, Ponderato N, Duchi R & Lazzari G 2003 Pregnancy: a cloned horse born to its dam twin. Nature 424 635. (https://doi.org/10.1038/424635a)

    • Search Google Scholar
    • Export Citation
  • Gasparrini B, Gao S, Ainslie A, Fletcher J, Mcgarry M, Ritchie WA, Springbett AJ, Overstrom EW, Wilmut I & De Sousa PA 2003 Cloned mice derived from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation. Biology of Reproduction 68 12591266. (https://doi.org/10.1095/biolreprod.102.008730)

    • Search Google Scholar
    • Export Citation
  • Gomez MC, Pope CE, Giraldo A, Lyons LA, Harris RF, King AL, Cole A, Godke RA & Dresser BL 2004 Birth of African Wildcat cloned kittens born from domestic cats. Cloning and Stem Cells 6 247258. (https://doi.org/10.1089/clo.2004.6.247)

    • Search Google Scholar
    • Export Citation
  • Gurdon JB 1962 The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. Journal of Embryology and Experimental Morphology 10 622640. (https://doi.org/10.1242/dev.10.4.622)

    • Search Google Scholar
    • Export Citation
  • Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert KM, Palmiter RD & Brinster RL 1985 Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315 680683. (https://doi.org/10.1038/315680a0)

    • Search Google Scholar
    • Export Citation
  • Hashimoto M & Takemoto T 2015 Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Scientific Reports 5 11315. (https://doi.org/10.1038/srep11315)

    • Search Google Scholar
    • Export Citation
  • Hill JR 2014 Incidence of abnormal offspring from cloning and other assisted reproductive technologies. Annual Review of Animal Biosciences 2 307321. (https://doi.org/10.1146/annurev-animal-022513-114109)

    • Search Google Scholar
    • Export Citation
  • Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, Hwang WS, Hossein MS, Kim JJ, Shin NS & Kang SK 2007 Endangered wolves cloned from adult somatic cells. Cloning and Stem Cells 9 130137. (https://doi.org/10.1089/clo.2006.0034)

    • Search Google Scholar
    • Export Citation
  • King TJ & Briggs R 1955 Changes in the nuclei of differentiating gastrula cells, as demonstrated by nuclear transplantation. PNAS 41 321325. (https://doi.org/10.1073/pnas.41.5.321)

    • Search Google Scholar
    • Export Citation
  • Klymiuk N, Blutke A, Graf A, Krause S, Burkhardt K, Wuensch A, Krebs S, Kessler B, Zakhartchenko V & Kurome M 2013 Dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle. Human Molecular Genetics 22 43684382. (https://doi.org/10.1093/hmg/ddt287)

    • Search Google Scholar
    • Export Citation
  • Koller BH & Smithies O 1992 Altering genes in animals by gene targeting. Annual Review of Immunology 10 705730. (https://doi.org/10.1146/annurev.iy.10.040192.003421)

    • Search Google Scholar
    • Export Citation
  • Kubota H, Avarbock MR & Brinster RL 2004 Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. PNAS 101 1648916494. (https://doi.org/10.1073/pnas.0407063101)

    • Search Google Scholar
    • Export Citation
  • Laible G, Wei J & Wagner S 2015 Improving livestock for agriculture - technological progress from random transgenesis to precision genome editing heralds a new era. Biotechnology Journal 10 109120. (https://doi.org/10.1002/biot.201400193)

    • Search Google Scholar
    • Export Citation
  • Lanza RP, Cibelli JB, Diaz F, Moraes CT, Farin PW, Farin CE, Hammer CJ, West MD & Damiani P 2000 Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer. Cloning 2 7990. (https://doi.org/10.1089/152045500436104)

    • Search Google Scholar
    • Export Citation
  • Lee BC, Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, Hossein MS, Kim JJ, Kang SK & Schatten G 2005 Dogs cloned from adult somatic cells. Nature 436 641. (https://doi.org/10.1038/436641a)

    • Search Google Scholar
    • Export Citation
  • Liu Z, Cai Y, Wang Y, Nie Y, Zhang C, Xu Y, Zhang X, Lu Y, Wang Z & Poo M 2018 Cloning of macaque monkeys by somatic cell nuclear transfer. Cell 174 245. (https://doi.org/10.1016/j.cell.2018.01.036)

    • Search Google Scholar
    • Export Citation
  • Mak IW, Evaniew N & Ghert M 2014 Lost in translation: animal models and clinical trials in cancer treatment. American Journal of Translational Research 6 114118.

    • Search Google Scholar
    • Export Citation
  • Matoba S & Zhang Y 2018 Somatic cell nuclear transfer reprogramming: mechanisms and applications. Cell Stem Cell 23 471485. (https://doi.org/10.1016/j.stem.2018.06.018)

    • Search Google Scholar
    • Export Citation
  • Mccreath KJ, Howcroft J, Campbell KH, Colman A, Schnieke AE & Kind AJ 2000 Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405 10661069. (https://doi.org/10.1038/35016604)

    • Search Google Scholar
    • Export Citation
  • Mcgrath J & Solter D 1983 Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science 220 13001302. (https://doi.org/10.1126/science.6857250)

    • Search Google Scholar
    • Export Citation
  • Meissner A & Jaenisch R 2006 Mammalian nuclear transfer. Developmental Dynamics 235 24602469. (https://doi.org/10.1002/dvdy.20915)

  • Nagy A, Gocza E, Diaz EM, Prideaux VR, Ivanyi E, Markkula M & Rossant J 1990 Embryonic stem cells alone are able to support fetal development in the mouse. Development 110 815821. (https://doi.org/10.1242/dev.110.3.815)

    • Search Google Scholar
    • Export Citation
  • Nambiar TS, Billon P, Diedenhofen G, Hayward SB, Taglialatela A, Cai K, Huang JW, Leuzzi G, Cuella-Martin R & Palacios A 2019 Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant. Nature Communications 10 3395. (https://doi.org/10.1038/s41467-019-11105-z)

    • Search Google Scholar
    • Export Citation
  • Niu D, Wei HJ, Lin L, George H, Wang T, Lee IH, Zhao HY, Wang Y, Kan Y & Shrock E 2017 Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science 357 13031307. (https://doi.org/10.1126/science.aan4187)

    • Search Google Scholar
    • Export Citation
  • Nour MS & Takahashi Y 1999 Preparation of young preactivated oocytes with high enucleation efficiency for bovine nuclear transfer. Theriogenology 51 661666. (https://doi.org/10.1016/s0093-691x(9900004-7)

    • Search Google Scholar
    • Export Citation
  • Ogura A, Inoue K & Wakayama T 2013 Recent advancements in cloning by somatic cell nuclear transfer. Philosophical Transactions of the Royal Society of London: Series B, Biological Sciences 368 20110329. (https://doi.org/10.1098/rstb.2011.0329)

    • Search Google Scholar
    • Export Citation
  • Perleberg C, Kind A & Schnieke A 2018 Genetically engineered pigs as model s for human disease. Disease Models and Mechanisms 11 dmm030783. (https://doi.org/10.1242/dmm.030783)

    • Search Google Scholar
    • Export Citation
  • Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S & Ayares DL 2000 Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407 8690. (https://doi.org/10.1038/35024082)

    • Search Google Scholar
    • Export Citation
  • Prather RS 2000 Cloning. Pigs is pigs. Science 289 18861887. (https://doi.org/10.1126/science.289.5486.1886)

  • Prather RS, Barnes FL, Sims MM, Robl JM, Eyestone WH & First NL 1987 Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte. Biology of Reproduction 37 859866. (https://doi.org/10.1095/biolreprod37.4.859)

    • Search Google Scholar
    • Export Citation
  • Prather RS, Sims MM & First NL 1989 Nuclear transplantation in early pig embryos. Biology of Reproduction 41 414418. (https://doi.org/10.1095/biolreprod41.3.414)

    • Search Google Scholar
    • Export Citation
  • Prather RS, Whitworth KM, Schommer SK & Wells KD 2017 Genetic engineering alveolar macrophages for host resistance to PRRSV. Veterinary Microbiology 209 124129. (https://doi.org/10.1016/j.vetmic.2017.01.036)

    • Search Google Scholar
    • Export Citation
  • Reichenbach M, Lim T, Reichenbach HD, Guengoer T, Habermann FA, Matthiesen M, Hofmann A, Weber F, Zerbe H & Grupp T 2010 Germ-line transmission of lentiviral PGK-EGFP integrants in transgenic cattle: new perspectives for experimental embryology. Transgenic Research 19 549556. (https://doi.org/10.1007/s11248-009-9333-5)

    • Search Google Scholar
    • Export Citation
  • Rogers CS 2016 Genetically engineered livestock for biomedical models. Transgenic Research 25 345359. (https://doi.org/10.1007/s11248-016-9928-6)

    • Search Google Scholar
    • Export Citation
  • Rogers CS, Abraham WM, Brogden KA, Engelhardt JF, Fisher JT, Mccray PB Jr, Mclennan G, Meyerholz DK, Namati E & Ostedgaard LS 2008 The porcine lung as a potential model for cystic fibrosis. American Journal of Physiology: Lung Cellular and Molecular Physiology 295 L240L263. (https://doi.org/10.1152/ajplung.90203.2008)

    • Search Google Scholar
    • Export Citation
  • Ruan J, Xu J, Chen-Tsai RY & Li K 2017 Genome editing in livestock: are we ready for a revolution in animal breeding industry? Transgenic Research 26 715726. (https://doi.org/10.1007/s11248-017-0049-7)

    • Search Google Scholar
    • Export Citation
  • Ryu J, Prather RS & Lee K 2018 Use of gene-editing technology to introduce targeted modifications in pigs. Journal of Animal Science and Biotechnology 9 5. (https://doi.org/10.1186/s40104-017-0228-7)

    • Search Google Scholar
    • Export Citation
  • Sander JD & Joung JK 2014 CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32 347355. (https://doi.org/10.1038/nbt.2842)

    • Search Google Scholar
    • Export Citation
  • Sayaka W, Satoshi K, Van Thuan N, Hiroshi O, Takafusa H, Eiji M, Thuy BH, Masashi M & Teruhiko W 2008 Effect of volume of oocyte cytoplasm on embryo development after parthenogenetic activation, intracytoplasmic sperm injection, or somatic cell nuclear transfer. Zygote 16 211222. (https://doi.org/10.1017/S0967199408004620)

    • Search Google Scholar
    • Export Citation
  • Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M, Wilmut I, Colman A & Campbell KH 1997 Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278 21302133. (https://doi.org/10.1126/science.278.5346.2130)

    • Search Google Scholar
    • Export Citation
  • Sherkow JS & Greely HT 2013 Genomics. What if extinction is not forever? Science 340 3233. (https://doi.org/10.1126/science.1236965)

  • Spemann H 1938 Embryonic Development and Induction. New Haven, London: Yale University Press; H Milford: Oxford University Press.

  • Steinborn R, Schinogl P, Zakhartchenko V, Achmann R, Schernthaner W, Stojkovic M, Wolf E, Muller M & Brem G 2000 Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning. Nature Genetics 25 255257. (https://doi.org/10.1038/77000)

    • Search Google Scholar
    • Export Citation
  • Stice SL & Robl JM 1988 Nuclear reprogramming in nuclear transplant rabbit embryos. Biology of Reproduction 39 657664. (https://doi.org/10.1095/biolreprod39.3.657)

    • Search Google Scholar
    • Export Citation
  • Tachibana M, Amato P, Sparman M, Gutierrez NM, Tippner-Hedges R, Ma H, Kang E, Fulati A, Lee HS & Sritanaudomchai H 2013 Human embryonic stem cells derived by somatic cell nuclear transfer. Cell 153 12281238. (https://doi.org/10.1016/j.cell.2013.05.006)

    • Search Google Scholar
    • Export Citation
  • Takahashi K & Yamanaka S 2006 Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126 663676. (https://doi.org/10.1016/j.cell.2006.07.024)

    • Search Google Scholar
    • Export Citation
  • Tecirlioglu RT, Cooney MA, Lewis IM, Korfiatis NA, Hodgson R, Ruddock NT, Vajta G, Downie S, Trounson AO & Holland MK 2005 Comparison of two approaches to nuclear transfer in the bovine: hand-made cloning with modifications and the conventional nuclear transfer technique. Reproduction, Fertility, and Development 17 573585. (https://doi.org/10.1071/rd04122)

    • Search Google Scholar
    • Export Citation
  • Vajta G, Lewis IM, Trounson AO, Purup S, Maddox-Hyttel P, Schmidt M, Pedersen HG, Greve T & Callesen H 2003 Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro. Biology of Reproduction 68 571578. (https://doi.org/10.1095/biolreprod.102.008771)

    • Search Google Scholar
    • Export Citation
  • Van Der Auwera I & D'hooghe T 2001 Superovulation of female mice delays embryonic and fetal development. Human Reproduction 16 12371243. (https://doi.org/10.1093/humrep/16.6.1237)

    • Search Google Scholar
    • Export Citation
  • Wakayama S, Ohta H, Hikichi T, Mizutani E, Iwaki T, Kanagawa O & Wakayama T 2008 Production of healthy cloned mice from bodies frozen at -20 degrees C for 16 years. PNAS 105 1731817322. (https://doi.org/10.1073/pnas.0806166105)

    • Search Google Scholar
    • Export Citation
  • Wakayama T, Perry AC, Zuccotti M, Johnson KR & Yanagimachi R 1998 Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394 369374. (https://doi.org/10.1038/28615)

    • Search Google Scholar
    • Export Citation
  • Weismann A, Parker WN & Rōnníeldt H 1893 The Germ-Plasm: a Theory of Heredity. New York: C. Scribner's Sons.

  • Willadsen SM 1986 Nuclear transplantation in sheep embryos. Nature 320 6365. (https://doi.org/10.1038/320063a0)

  • Wilmut I, Schnieke AE, Mcwhir J, Kind AJ & Campbell KH 1997 Viable offspring derived from fetal and adult mammalian cells. Nature 385 810813. (https://doi.org/10.1038/385810a0)

    • Search Google Scholar
    • Export Citation
  • Wolf E, Schernthaner W, Zakhartchenko V, Prelle K, Stojkovic M & Brem G 2000 Transgenic technology in farm animals--progress and perspectives. Experimental Physiology 85 615625. (https://doi.org/10.1111/j.1469-445X.2000.02110.x)

    • Search Google Scholar
    • Export Citation
  • Wolf E, Braun-Reichhart C, Streckel E & Renner S 2014 Genetically engineered pig models for diabetes research. Transgenic Research 23 2738. (https://doi.org/10.1007/s11248-013-9755-y)

    • Search Google Scholar
    • Export Citation
  • Young LE, Sinclair KD & Wilmut I 1998 Large offspring syndrome in cattle and sheep. Reviews of Reproduction 3 155163. (https://doi.org/10.1530/ror.0.0030155)

    • Search Google Scholar
    • Export Citation
  • Yuan Y & Krisher RL 2012 In vitro maturation (IVM) of porcine oocytes. Methods in Molecular Biology 825 183198. (https://doi.org/10.1007/978-1-61779-436-0_14)

    • Search Google Scholar
    • Export Citation
  • Zhang J, Liu H, Luo S, Lu Z, Chavez-Badiola A, Liu Z, Yang M, Merhi Z, Silber SJ & Munne S 2017 Live birth derived from oocyte spindle transfer to prevent mitochondrial disease. Reproductive Biomedicine Online 34 361368. (https://doi.org/10.1016/j.rbmo.2017.01.013)

    • Search Google Scholar
    • Export Citation
  • Zhang XH, Tee LY, Wang XG, Huang QS & Yang SH 2015 Off-target effects in CRISPR/Cas9-mediated genome engineering. Molecular Therapy: Nucleic Acids 4 e264. (https://doi.org/10.1038/mtna.2015.37)

    • Search Google Scholar
    • Export Citation
  • Zhao S, Jiang E, Chen S, Gu Y, Shangguan AJ, Lv T, Luo L & Yu Z 2016 PiggyBac transposon vectors: the tools of the human gene encoding. Translational Lung Cancer Research 5 120125. (https://doi.org/10.3978/j.issn.2218-6751.2016.01.05)

    • Search Google Scholar
    • Export Citation

 

  • Collapse
  • Expand

     An official journal of

    Society for Reproduction and Fertility

 

  • View in gallery
    Figure 1

    The development of SCNT and related technology.

  • View in gallery
    Figure 2

    Timeline of cloned mammalian species over the last 25 years.

  • View in gallery
    Figure 3

    The toolkit for the generation of genetically modified livestock. (A) Pronuclear DNA microinjection leads to random transgene integration but does not facilitate gene targeting. Viral vectors and transposons can be used to increase the frequency of transgenesis. (B) Cytoplasmic microinjection of site-specific endonucleases facilitates targeted genome engineering directly in early embryos. (C) Genetically engineered somatic cells selected for the presence of the desired modification. Single cells are inserted into the perivitelline space of enucleated oocytes, followed by cell fusion and activation to generate reconstructed embryos for embryo transfer.

  • Akagi S, Adachi N, Matsukawa K, Kubo M & Takahashi S 2003 Developmental potential of bovine nuclear transfer embryos and postnatal survival rate of cloned calves produced by two different timings of fusion and activation. Molecular Reproduction and Development 66 264272. (https://doi.org/10.1002/mrd.10352)

    • Search Google Scholar
    • Export Citation
  • Anderson KR, Haeussler M, Watanabe C, Janakiraman V, Lund J, Modrusan Z, Stinson J, Bei Q, Buechler A & Yu C 2018 CRISPR off-target analysis in genetically engineered rats and mice. Nature Methods 15 512514. (https://doi.org/10.1038/s41592-018-0011-5)

    • Search Google Scholar
    • Export Citation
  • Bogliotti YS, Wu J, Vilarino M, Okamura D, Soto DA, Zhong C, Sakurai M, Sampaio RV, Suzuki K & Izpisua Belmonte JC 2018 Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. PNAS 115 20902095. (https://doi.org/10.1073/pnas.1716161115)

    • Search Google Scholar
    • Export Citation
  • Briggs R & King TJ 1952 Transplantation of living nuclei from blastula cells into enucleated frogs' eggs. PNAS 38 455463. (https://doi.org/10.1073/pnas.38.5.455)

    • Search Google Scholar
    • Export Citation
  • Brinster RL, Sandgren EP, Behringer RR & Palmiter RD 1989 No simple solution for making transgenic mice. Cell 59 239241. (https://doi.org/10.1016/0092-8674(8990282-1)

    • Search Google Scholar
    • Export Citation
  • Campbell KH, Mcwhir J, Ritchie WA & Wilmut I 1996 Sheep cloned by nuclear transfer from a cultured cell line. Nature 380 6466. (https://doi.org/10.1038/380064a0)

    • Search Google Scholar
    • Export Citation
  • Capecchi MR 1989 aAltering the genome by homologous recombination. Science 244 12881292. (https://doi.org/10.1126/science.2660260)

  • Capecchi MR 1989 bThe new mouse genetics: altering the genome by gene targeting. Trends in Genetics 5 7076. (https://doi.org/10.1016/0168-9525(8990029-2)

    • Search Google Scholar
    • Export Citation
  • Carbery ID, Ji D, Harrington A, Brown V, Weinstein EJ, Liaw L & Cui X 2010 Targeted genome modification in mice using zinc-finger nucleases. Genetics 186 451459. (https://doi.org/10.1534/genetics.110.117002)

    • Search Google Scholar
    • Export Citation
  • Carlson DF, Lancto CA, Zang B, Kim ES, Walton M, Oldeschulte D, Seabury C, Sonstegard TS & Fahrenkrug SC 2016 Production of hornless dairy cattle from genome-edited cell lines. Nature Biotechnology 34 479481. (https://doi.org/10.1038/nbt.3560)

    • Search Google Scholar
    • Export Citation
  • Cibelli JB & Gurdon JB 2018 Custom-made oocytes to clone non-human Primates. Cell 172 647649. (https://doi.org/10.1016/j.cell.2018.01.030)

  • Ciccarelli M, Giassetti MI, Miao D, Oatley MJ, Robbins C, Lopez-Biladeau B, Waqas MS, Tibary A, Whitelaw B & Lillico S 2020 Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males. PNAS 117 2419524204. (https://doi.org/10.1073/pnas.2010102117)

    • Search Google Scholar
    • Export Citation
  • Dominko T, Chan A, Simerly C, Luetjens CM, Hewitson L, Martinovich C & Schatten G 2000 Dynamic imaging of the metaphase II spindle and maternal chromosomesin bovine oocytes: implications for enucleation efficiency verification, avoidanceof parthenogenesis, and successful embryogenesis. Biology of Reproduction 62 150154. (https://doi.org/10.1095/biolreprod62.1.150)

    • Search Google Scholar
    • Export Citation
  • Fischer K, Kraner-Scheiber S, Petersen B, Rieblinger B, Buermann A, Flisikowska T, Flisikowski K, Christan S, Edlinger M & Baars W 2016 Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing. Scientific Reports 6 29081. (https://doi.org/10.1038/srep29081)

    • Search Google Scholar
    • Export Citation
  • Fischer K, Rieblinger B, Hein R, Sfriso R, Zuber J, Fischer A, Klinger B, Liang W, Flisikowski K & Kurome M 2019 Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2. Xenotransplantation 27 e12560. (https://doi.org/10.1111/xen.12560)

    • Search Google Scholar
    • Export Citation
  • Fleming TP, Kwong WY, Porter R, Ursell E, Fesenko I, Wilkins A, Miller DJ, Watkins AJ & Eckert JJ 2004 The embryo and its future. Biology of Reproduction 71 10461054. (https://doi.org/10.1095/biolreprod.104.030957)

    • Search Google Scholar
    • Export Citation
  • Flisikowska T, Kind A & Schnieke A 2013 The new pig on the block: modelling cancer in pigs. Transgenic Research 22 673680. (https://doi.org/10.1007/s11248-013-9720-9)

    • Search Google Scholar
    • Export Citation
  • Folch J, Cocero MJ, Chesne P, Alabart JL, Dominguez V, Cognie Y, Roche A, Fernandez-Arias A, Marti JI & Sanchez P 2009 First birth of an animal from an extinct subspecies (Capra pyrenaica pyrenaica) by cloning. Theriogenology 71 10261034. (https://doi.org/10.1016/j.theriogenology.2008.11.005)

    • Search Google Scholar
    • Export Citation
  • Gaj T, Gersbach CA & Barbas CF 3RD 2013 ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology 31 397405. (https://doi.org/10.1016/j.tibtech.2013.04.004)

    • Search Google Scholar
    • Export Citation
  • Galli C, Lagutina I, Crotti G, Colleoni S, Turini P, Ponderato N, Duchi R & Lazzari G 2003 Pregnancy: a cloned horse born to its dam twin. Nature 424 635. (https://doi.org/10.1038/424635a)

    • Search Google Scholar
    • Export Citation
  • Gasparrini B, Gao S, Ainslie A, Fletcher J, Mcgarry M, Ritchie WA, Springbett AJ, Overstrom EW, Wilmut I & De Sousa PA 2003 Cloned mice derived from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation. Biology of Reproduction 68 12591266. (https://doi.org/10.1095/biolreprod.102.008730)

    • Search Google Scholar
    • Export Citation
  • Gomez MC, Pope CE, Giraldo A, Lyons LA, Harris RF, King AL, Cole A, Godke RA & Dresser BL 2004 Birth of African Wildcat cloned kittens born from domestic cats. Cloning and Stem Cells 6 247258. (https://doi.org/10.1089/clo.2004.6.247)

    • Search Google Scholar
    • Export Citation
  • Gurdon JB 1962 The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. Journal of Embryology and Experimental Morphology 10 622640. (https://doi.org/10.1242/dev.10.4.622)

    • Search Google Scholar
    • Export Citation
  • Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert KM, Palmiter RD & Brinster RL 1985 Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315 680683. (https://doi.org/10.1038/315680a0)

    • Search Google Scholar
    • Export Citation
  • Hashimoto M & Takemoto T 2015 Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Scientific Reports 5 11315. (https://doi.org/10.1038/srep11315)

    • Search Google Scholar
    • Export Citation
  • Hill JR 2014 Incidence of abnormal offspring from cloning and other assisted reproductive technologies. Annual Review of Animal Biosciences 2 307321. (https://doi.org/10.1146/annurev-animal-022513-114109)

    • Search Google Scholar
    • Export Citation
  • Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, Hwang WS, Hossein MS, Kim JJ, Shin NS & Kang SK 2007 Endangered wolves cloned from adult somatic cells. Cloning and Stem Cells 9 130137. (https://doi.org/10.1089/clo.2006.0034)

    • Search Google Scholar
    • Export Citation
  • King TJ & Briggs R 1955 Changes in the nuclei of differentiating gastrula cells, as demonstrated by nuclear transplantation. PNAS 41 321325. (https://doi.org/10.1073/pnas.41.5.321)

    • Search Google Scholar
    • Export Citation
  • Klymiuk N, Blutke A, Graf A, Krause S, Burkhardt K, Wuensch A, Krebs S, Kessler B, Zakhartchenko V & Kurome M 2013 Dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle. Human Molecular Genetics 22 43684382. (https://doi.org/10.1093/hmg/ddt287)

    • Search Google Scholar
    • Export Citation
  • Koller BH & Smithies O 1992 Altering genes in animals by gene targeting. Annual Review of Immunology 10 705730. (https://doi.org/10.1146/annurev.iy.10.040192.003421)

    • Search Google Scholar
    • Export Citation
  • Kubota H, Avarbock MR & Brinster RL 2004 Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. PNAS 101 1648916494. (https://doi.org/10.1073/pnas.0407063101)

    • Search Google Scholar
    • Export Citation
  • Laible G, Wei J & Wagner S 2015 Improving livestock for agriculture - technological progress from random transgenesis to precision genome editing heralds a new era. Biotechnology Journal 10 109120. (https://doi.org/10.1002/biot.201400193)

    • Search Google Scholar
    • Export Citation
  • Lanza RP, Cibelli JB, Diaz F, Moraes CT, Farin PW, Farin CE, Hammer CJ, West MD & Damiani P 2000 Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer. Cloning 2 7990. (https://doi.org/10.1089/152045500436104)

    • Search Google Scholar
    • Export Citation
  • Lee BC, Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, Hossein MS, Kim JJ, Kang SK & Schatten G 2005 Dogs cloned from adult somatic cells. Nature 436 641. (https://doi.org/10.1038/436641a)

    • Search Google Scholar
    • Export Citation
  • Liu Z, Cai Y, Wang Y, Nie Y, Zhang C, Xu Y, Zhang X, Lu Y, Wang Z & Poo M 2018 Cloning of macaque monkeys by somatic cell nuclear transfer. Cell 174 245. (https://doi.org/10.1016/j.cell.2018.01.036)

    • Search Google Scholar
    • Export Citation
  • Mak IW, Evaniew N & Ghert M 2014 Lost in translation: animal models and clinical trials in cancer treatment. American Journal of Translational Research 6 114118.

    • Search Google Scholar
    • Export Citation
  • Matoba S & Zhang Y 2018 Somatic cell nuclear transfer reprogramming: mechanisms and applications. Cell Stem Cell 23 471485. (https://doi.org/10.1016/j.stem.2018.06.018)

    • Search Google Scholar
    • Export Citation
  • Mccreath KJ, Howcroft J, Campbell KH, Colman A, Schnieke AE & Kind AJ 2000 Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405 10661069. (https://doi.org/10.1038/35016604)

    • Search Google Scholar
    • Export Citation
  • Mcgrath J & Solter D 1983 Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science 220 13001302. (https://doi.org/10.1126/science.6857250)

    • Search Google Scholar
    • Export Citation
  • Meissner A & Jaenisch R 2006 Mammalian nuclear transfer. Developmental Dynamics 235 24602469. (https://doi.org/10.1002/dvdy.20915)

  • Nagy A, Gocza E, Diaz EM, Prideaux VR, Ivanyi E, Markkula M & Rossant J 1990 Embryonic stem cells alone are able to support fetal development in the mouse. Development 110 815821. (https://doi.org/10.1242/dev.110.3.815)

    • Search Google Scholar
    • Export Citation
  • Nambiar TS, Billon P, Diedenhofen G, Hayward SB, Taglialatela A, Cai K, Huang JW, Leuzzi G, Cuella-Martin R & Palacios A 2019 Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant. Nature Communications 10 3395. (https://doi.org/10.1038/s41467-019-11105-z)

    • Search Google Scholar
    • Export Citation
  • Niu D, Wei HJ, Lin L, George H, Wang T, Lee IH, Zhao HY, Wang Y, Kan Y & Shrock E 2017 Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science 357 13031307. (https://doi.org/10.1126/science.aan4187)

    • Search Google Scholar
    • Export Citation
  • Nour MS & Takahashi Y 1999 Preparation of young preactivated oocytes with high enucleation efficiency for bovine nuclear transfer. Theriogenology 51 661666. (https://doi.org/10.1016/s0093-691x(9900004-7)

    • Search Google Scholar
    • Export Citation
  • Ogura A, Inoue K & Wakayama T 2013 Recent advancements in cloning by somatic cell nuclear transfer. Philosophical Transactions of the Royal Society of London: Series B, Biological Sciences 368 20110329. (https://doi.org/10.1098/rstb.2011.0329)

    • Search Google Scholar
    • Export Citation
  • Perleberg C, Kind A & Schnieke A 2018 Genetically engineered pigs as model s for human disease. Disease Models and Mechanisms 11 dmm030783. (https://doi.org/10.1242/dmm.030783)

    • Search Google Scholar
    • Export Citation
  • Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S & Ayares DL 2000 Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407 8690. (https://doi.org/10.1038/35024082)

    • Search Google Scholar
    • Export Citation
  • Prather RS 2000 Cloning. Pigs is pigs. Science 289 18861887. (https://doi.org/10.1126/science.289.5486.1886)

  • Prather RS, Barnes FL, Sims MM, Robl JM, Eyestone WH & First NL 1987 Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte. Biology of Reproduction 37 859866. (https://doi.org/10.1095/biolreprod37.4.859)

    • Search Google Scholar
    • Export Citation
  • Prather RS, Sims MM & First NL 1989 Nuclear transplantation in early pig embryos. Biology of Reproduction 41 414418. (https://doi.org/10.1095/biolreprod41.3.414)

    • Search Google Scholar
    • Export Citation
  • Prather RS, Whitworth KM, Schommer SK & Wells KD 2017 Genetic engineering alveolar macrophages for host resistance to PRRSV. Veterinary Microbiology 209 124129. (https://doi.org/10.1016/j.vetmic.2017.01.036)

    • Search Google Scholar
    • Export Citation
  • Reichenbach M, Lim T, Reichenbach HD, Guengoer T, Habermann FA, Matthiesen M, Hofmann A, Weber F, Zerbe H & Grupp T 2010 Germ-line transmission of lentiviral PGK-EGFP integrants in transgenic cattle: new perspectives for experimental embryology. Transgenic Research 19 549556. (https://doi.org/10.1007/s11248-009-9333-5)

    • Search Google Scholar
    • Export Citation
  • Rogers CS 2016 Genetically engineered livestock for biomedical models. Transgenic Research 25 345359. (https://doi.org/10.1007/s11248-016-9928-6)

    • Search Google Scholar
    • Export Citation
  • Rogers CS, Abraham WM, Brogden KA, Engelhardt JF, Fisher JT, Mccray PB Jr, Mclennan G, Meyerholz DK, Namati E & Ostedgaard LS 2008 The porcine lung as a potential model for cystic fibrosis. American Journal of Physiology: Lung Cellular and Molecular Physiology 295 L240L263. (https://doi.org/10.1152/ajplung.90203.2008)

    • Search Google Scholar
    • Export Citation
  • Ruan J, Xu J, Chen-Tsai RY & Li K 2017 Genome editing in livestock: are we ready for a revolution in animal breeding industry? Transgenic Research 26 715726. (https://doi.org/10.1007/s11248-017-0049-7)

    • Search Google Scholar
    • Export Citation
  • Ryu J, Prather RS & Lee K 2018 Use of gene-editing technology to introduce targeted modifications in pigs. Journal of Animal Science and Biotechnology 9 5. (https://doi.org/10.1186/s40104-017-0228-7)

    • Search Google Scholar
    • Export Citation
  • Sander JD & Joung JK 2014 CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32 347355. (https://doi.org/10.1038/nbt.2842)

    • Search Google Scholar
    • Export Citation
  • Sayaka W, Satoshi K, Van Thuan N, Hiroshi O, Takafusa H, Eiji M, Thuy BH, Masashi M & Teruhiko W 2008 Effect of volume of oocyte cytoplasm on embryo development after parthenogenetic activation, intracytoplasmic sperm injection, or somatic cell nuclear transfer. Zygote 16 211222. (https://doi.org/10.1017/S0967199408004620)

    • Search Google Scholar
    • Export Citation
  • Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M, Wilmut I, Colman A & Campbell KH 1997 Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 278 21302133. (https://doi.org/10.1126/science.278.5346.2130)

    • Search Google Scholar
    • Export Citation
  • Sherkow JS & Greely HT 2013 Genomics. What if extinction is not forever? Science 340 3233. (https://doi.org/10.1126/science.1236965)

  • Spemann H 1938 Embryonic Development and Induction. New Haven, London: Yale University Press; H Milford: Oxford University Press.

  • Steinborn R, Schinogl P, Zakhartchenko V, Achmann R, Schernthaner W, Stojkovic M, Wolf E, Muller M & Brem G 2000 Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning. Nature Genetics 25 255257. (https://doi.org/10.1038/77000)

    • Search Google Scholar
    • Export Citation
  • Stice SL & Robl JM 1988 Nuclear reprogramming in nuclear transplant rabbit embryos. Biology of Reproduction 39 657664. (https://doi.org/10.1095/biolreprod39.3.657)

    • Search Google Scholar
    • Export Citation
  • Tachibana M, Amato P, Sparman M, Gutierrez NM, Tippner-Hedges R, Ma H, Kang E, Fulati A, Lee HS & Sritanaudomchai H 2013 Human embryonic stem cells derived by somatic cell nuclear transfer. Cell 153 12281238. (https://doi.org/10.1016/j.cell.2013.05.006)

    • Search Google Scholar
    • Export Citation
  • Takahashi K & Yamanaka S 2006 Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126 663676. (https://doi.org/10.1016/j.cell.2006.07.024)

    • Search Google Scholar
    • Export Citation
  • Tecirlioglu RT, Cooney MA, Lewis IM, Korfiatis NA, Hodgson R, Ruddock NT, Vajta G, Downie S, Trounson AO & Holland MK 2005 Comparison of two approaches to nuclear transfer in the bovine: hand-made cloning with modifications and the conventional nuclear transfer technique. Reproduction, Fertility, and Development 17 573585. (https://doi.org/10.1071/rd04122)

    • Search Google Scholar
    • Export Citation
  • Vajta G, Lewis IM, Trounson AO, Purup S, Maddox-Hyttel P, Schmidt M, Pedersen HG, Greve T & Callesen H 2003 Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro. Biology of Reproduction 68 571578. (https://doi.org/10.1095/biolreprod.102.008771)

    • Search Google Scholar
    • Export Citation
  • Van Der Auwera I & D'hooghe T 2001 Superovulation of female mice delays embryonic and fetal development. Human Reproduction 16 12371243. (https://doi.org/10.1093/humrep/16.6.1237)

    • Search Google Scholar
    • Export Citation
  • Wakayama S, Ohta H, Hikichi T, Mizutani E, Iwaki T, Kanagawa O & Wakayama T 2008 Production of healthy cloned mice from bodies frozen at -20 degrees C for 16 years. PNAS 105 1731817322. (https://doi.org/10.1073/pnas.0806166105)

    • Search Google Scholar
    • Export Citation
  • Wakayama T, Perry AC, Zuccotti M, Johnson KR & Yanagimachi R 1998 Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394 369374. (https://doi.org/10.1038/28615)

    • Search Google Scholar
    • Export Citation
  • Weismann A, Parker WN & Rōnníeldt H 1893 The Germ-Plasm: a Theory of Heredity. New York: C. Scribner's Sons.

  • Willadsen SM 1986 Nuclear transplantation in sheep embryos. Nature 320 6365. (https://doi.org/10.1038/320063a0)

  • Wilmut I, Schnieke AE, Mcwhir J, Kind AJ & Campbell KH 1997 Viable offspring derived from fetal and adult mammalian cells. Nature 385 810813. (https://doi.org/10.1038/385810a0)

    • Search Google Scholar
    • Export Citation
  • Wolf E, Schernthaner W, Zakhartchenko V, Prelle K, Stojkovic M & Brem G 2000 Transgenic technology in farm animals--progress and perspectives. Experimental Physiology 85 615625. (https://doi.org/10.1111/j.1469-445X.2000.02110.x)

    • Search Google Scholar
    • Export Citation
  • Wolf E, Braun-Reichhart C, Streckel E & Renner S 2014 Genetically engineered pig models for diabetes research. Transgenic Research 23 2738. (https://doi.org/10.1007/s11248-013-9755-y)

    • Search Google Scholar
    • Export Citation
  • Young LE, Sinclair KD & Wilmut I 1998 Large offspring syndrome in cattle and sheep. Reviews of Reproduction 3 155163. (https://doi.org/10.1530/ror.0.0030155)

    • Search Google Scholar
    • Export Citation
  • Yuan Y & Krisher RL 2012 In vitro maturation (IVM) of porcine oocytes. Methods in Molecular Biology 825 183198. (https://doi.org/10.1007/978-1-61779-436-0_14)

    • Search Google Scholar
    • Export Citation
  • Zhang J, Liu H, Luo S, Lu Z, Chavez-Badiola A, Liu Z, Yang M, Merhi Z, Silber SJ & Munne S 2017 Live birth derived from oocyte spindle transfer to prevent mitochondrial disease. Reproductive Biomedicine Online 34 361368. (https://doi.org/10.1016/j.rbmo.2017.01.013)

    • Search Google Scholar
    • Export Citation
  • Zhang XH, Tee LY, Wang XG, Huang QS & Yang SH 2015 Off-target effects in CRISPR/Cas9-mediated genome engineering. Molecular Therapy: Nucleic Acids 4 e264. (https://doi.org/10.1038/mtna.2015.37)

    • Search Google Scholar
    • Export Citation
  • Zhao S, Jiang E, Chen S, Gu Y, Shangguan AJ, Lv T, Luo L & Yu Z 2016 PiggyBac transposon vectors: the tools of the human gene encoding. Translational Lung Cancer Research 5 120125. (https://doi.org/10.3978/j.issn.2218-6751.2016.01.05)

    • Search Google Scholar
    • Export Citation