Breaking the final barrier in reproductive animal cloning: macaque monkeys cloned

Breaking the final barrier in reproductive animal cloning: macaque monkeys cloned Animal models are essential in research aiming to understand human biology and diseases, yet far too often clinical trials fail with drugs and therapeutic approaches that have been developed through, tested and built upon research in animal models. As species genetically the closest to humans, non-human primates are considered to be the best models available for studying human biology, modeling diseases and testing therapeutic strategies. However, because of genetic heterogeneity at the individual level, controlling phenotypic variabilities typically requires the analysis of a large number of animals, which is costly and raises potential ethical concerns. Now, Sun and colleagues from the Chinese Academy of Sciences have reported the cloning of macaque monkeys by somatic nuclear transfer (SCNT) using a genetically tractable and renewable source of macaque fetal skin fibroblasts [1], achieving a major milestone in reproductive animal cloning and launching a new era for basic and biomedical research. The cloning of animals by SCNT can be traced back to over half a century ago [2]. John Gurdon cloned a frog by injecting single intact nuclei from somatic cells from a Xenopus tadpole in 1958 [3], and Dizhou Tong (童第周,1902–1979) and colleagues cloned a goldfish by injecting the nucleus of a goldfish blastocyst into a goldfish egg in 1963 [4]. Probably the most famous cloned animal to date is a sheep named ‘Dolly’, the first mammal cloned from a somatic cell of an adult animal in 1997 [5]. Many other animals have been cloned from somatic cells, such as mice, cattle, cats, deer, dogs, horses, mules, oxen, rabbits and rats, suggesting that somatic cell nuclei from many species can be effectively reprogrammed in eggs to produce live offspring. However, one notable exception was cloning of non-human primates by SCNT; over the past decade, attempts by multiple laboratories across the world have failed. Rhesus monkeys were first cloned using blastomeres derived from in vitro fertilization-produced embryos as donors for nuclear transfer [6]. In addition, a rhesus macaque named ‘Tetra’ was created through ‘embryo splitting’, a process where the cells in an embryo are split at the eight-cell stage to create four identical two-cell embryos [7]. Because cloning by this approach takes place after the formation of an embryo, it is only possible to generate limited copies. Together, these results raised the biological question of whether somatic cell nuclei from non-human primates are intrinsically resistant to being reprogrammed in eggs to generate live and viable offspring. Working with fetal monkey fibroblasts, Sun's team started by optimizing many steps in the SCNT protocol [1]. The investigators’ experience was a key factor, such as the time to complete the entire procedure of SCNT. Perhaps most critically, the authors built on recent findings in other species showing that the epigenetic status of the donor nuclei could be a barrier to the proper development of SCNT embryos and, in particular, that expression of human histone H3K9me3 demethylase KDM4D/4A significantly improves the efficacy of both mouse and human SCNT [8,9]. Using transcriptomic analysis, they found that many genes are upregulated during the four- to eight-cell transition of SCNT embryos and that reactivation of some developmentally regulated pluripotency-associated genes requires the removal of the H3K9me3 epigenetic mark [1]. The authors then determined an optimal combination of the timing and concentration of H3K9me3 demethylase Kdm4d expression by mRNA injection and histone deacetylase inhibitor application using Trichostatin A. Using fetal skin fibroblasts from an aborted female cynomolgus monkey fetus as the source of nuclei, a total of 79 SCNT embryos between the two-cell to blastocyst stages were transferred to 21 cynomolgus female surrogates, leading to six pregnancies and two live births at full-term by caesarean section. These two baby monkeys, named ‘Zhongzhong’ and ‘Huahua’ remain healthy and growing (Fig. 1). The authors also performed similar experiments with adult cumulus cells of female monkeys as the donors. A total of 181 of SCNT embryos at similar stages were transferred to 42 surrogates, leading to 22 pregnancies and 2 live births by caesarean section, but both died within 30 h due to respiratory failure. It is not yet clear why adult somatic cells as a donor failed; potential reasons include epigenetic differences or telomere length between fetal and adult somatic cells. Importantly, genetic analysis of mitochondrial and nuclear DNAs confirmed the clonal origin of the four monkeys generated by the SCNT approach. Figure 1. View largeDownload slide An updated picture of ‘Zhongzhong’ (65 days old) and ‘Huahua’ (55 days old), two baby Macaque monkeys cloned by somatic cell nuclear transfer (Courtesy of Qiang Sun and Zhen Liu, Institute of Neuroscience, Chinese Academy of Sciences). Can you guess who is who? Figure 1. View largeDownload slide An updated picture of ‘Zhongzhong’ (65 days old) and ‘Huahua’ (55 days old), two baby Macaque monkeys cloned by somatic cell nuclear transfer (Courtesy of Qiang Sun and Zhen Liu, Institute of Neuroscience, Chinese Academy of Sciences). Can you guess who is who? This demonstration of the feasibility of reproductive cloning of macaque monkeys using somatic cell nuclei breaks a technical barrier and ushers in a new era of using non-human primates as experimental models. Because primate colonies normally require outbreeding to maintain genetic diversity for the health of the animals, the ability to generate a large number of genetically tractable and uniform non-human primates opens up countless opportunities. Reduced phenotypic variability will require much a smaller number of animals for a particular project. We can now generate a detailed reference database for these non-human primates on defined genetic backgrounds at the molecular, cellular, tissue and organ, and behavioral levels. Importantly, somatic cells, such as fetal skin fibroblasts, can be used for genome editing and screening, which is much more efficient than manipulating the embryos directly and avoids issues of mosaicism. Isogenic clones with modified genomes, such as knock-ins of reporters or human disease mutations, can be generated, phenotyped and compared to the reference database. These tools will not only be useful for increasing our understanding of basic biology and physiology, especially of primate-specific features, but will greatly facilitate biomedical discoveries and drug testing. For example, disease models can be used for discovery of early biomarkers for different disorders for diagnostics and for testing candidate therapeutic strategies. What are the challenges ahead? ‘Zhongzhong’ and ‘Huahua’ need to be closely monitored over the long-term for any signs of abnormality. Additional cloned monkeys with different genetic backgrounds are also needed for comparison. The current success provides a proof-of-principle case for the reprogramming competency of primate somatic nuclei, yet the efficacy of SCNT for non-human primates is still extremely low. With the conceptual barrier removed, it is likely that the technology will be rapidly improved. The dilemma is that the more efficient we become at SCNT cloning of monkeys, the closer it brings us to the ethical boundary of cloning humans. It is now time for an open discussion to establish guidelines, and perhaps international laws to prohibit even testing the possibility. Ethical guidelines are also needed for genome-editing in monkeys, given the possibility of knocking-in human-specific genes to achieve ‘humanized’ models, especially for the brain. In ‘Journey to the West’, a classic Chinese novel published in the 16th century, Monkey King, with a hair plucked from his body and a gentle blow, could produce unlimited copies of himself. While SCNT cloning of monkeys is not yet that simple in real life, this landmark study opens up a new chapter in the generation of non-human primate models for basic and translational research, and has the potential to accelerate the development of effective and safe new treatments for human diseases in the near future. REFERENCES 1. Liu Z , Cai Y , Wang Y et al. Cell 2018 ; 172 : 881 – 7.e7 . https://doi.org/10.1016/j.cell.2018.01.020 CrossRef Search ADS PubMed 2. Wilmut I , Bai Y , Taylor J . Phil Trans R Soc B 2015 ; 370 : 20140366 . https://doi.org/10.1098/rstb.2014.0366 CrossRef Search ADS PubMed 3. Gurdon JB , Elsdale TR , Fischberg M . Nature 1958 ; 182 : 64 – 5 . https://doi.org/10.1038/182064a0 CrossRef Search ADS PubMed 4. 童第周, 吴尚懃, 叶毓芬. 鱼类细胞核的移植. 科学通报 ; 1963 ; 14 : 60 – 1 . 5. Wilmut I , Schnieke AE , McWhir J et al. Nature 1997 ; 385 : 810 – 3 . https://doi.org/10.1038/385810a0 CrossRef Search ADS PubMed 6. Meng L , Ely JJ , Stouffer RL et al. Biol Reprod 1997 ; 57 : 454 – 9 . https://doi.org/10.1095/biolreprod57.2.454 CrossRef Search ADS PubMed 7. Chan AW , Dominko T , Luetjens CM et al. Science 2000 ; 287 : 317 – 9 . https://doi.org/10.1126/science.287.5451.317 CrossRef Search ADS PubMed 8. Antony J , Oback F , Chamley LW et al. Mol Cell Biol 2013 ; 33 : 974 – 83 . https://doi.org/10.1128/MCB.01014-12 CrossRef Search ADS PubMed 9. Chung YG , Matoba S , Liu Y et al. Cell Stem Cell 2015 ; 17 : 758 – 66 . https://doi.org/10.1016/j.stem.2015.10.001 CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png National Science Review Oxford University Press

Breaking the final barrier in reproductive animal cloning: macaque monkeys cloned

National Science Review , Volume Advance Article (3) – Feb 15, 2018

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd.
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2095-5138
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2053-714X
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10.1093/nsr/nwy028
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Abstract

Animal models are essential in research aiming to understand human biology and diseases, yet far too often clinical trials fail with drugs and therapeutic approaches that have been developed through, tested and built upon research in animal models. As species genetically the closest to humans, non-human primates are considered to be the best models available for studying human biology, modeling diseases and testing therapeutic strategies. However, because of genetic heterogeneity at the individual level, controlling phenotypic variabilities typically requires the analysis of a large number of animals, which is costly and raises potential ethical concerns. Now, Sun and colleagues from the Chinese Academy of Sciences have reported the cloning of macaque monkeys by somatic nuclear transfer (SCNT) using a genetically tractable and renewable source of macaque fetal skin fibroblasts [1], achieving a major milestone in reproductive animal cloning and launching a new era for basic and biomedical research. The cloning of animals by SCNT can be traced back to over half a century ago [2]. John Gurdon cloned a frog by injecting single intact nuclei from somatic cells from a Xenopus tadpole in 1958 [3], and Dizhou Tong (童第周,1902–1979) and colleagues cloned a goldfish by injecting the nucleus of a goldfish blastocyst into a goldfish egg in 1963 [4]. Probably the most famous cloned animal to date is a sheep named ‘Dolly’, the first mammal cloned from a somatic cell of an adult animal in 1997 [5]. Many other animals have been cloned from somatic cells, such as mice, cattle, cats, deer, dogs, horses, mules, oxen, rabbits and rats, suggesting that somatic cell nuclei from many species can be effectively reprogrammed in eggs to produce live offspring. However, one notable exception was cloning of non-human primates by SCNT; over the past decade, attempts by multiple laboratories across the world have failed. Rhesus monkeys were first cloned using blastomeres derived from in vitro fertilization-produced embryos as donors for nuclear transfer [6]. In addition, a rhesus macaque named ‘Tetra’ was created through ‘embryo splitting’, a process where the cells in an embryo are split at the eight-cell stage to create four identical two-cell embryos [7]. Because cloning by this approach takes place after the formation of an embryo, it is only possible to generate limited copies. Together, these results raised the biological question of whether somatic cell nuclei from non-human primates are intrinsically resistant to being reprogrammed in eggs to generate live and viable offspring. Working with fetal monkey fibroblasts, Sun's team started by optimizing many steps in the SCNT protocol [1]. The investigators’ experience was a key factor, such as the time to complete the entire procedure of SCNT. Perhaps most critically, the authors built on recent findings in other species showing that the epigenetic status of the donor nuclei could be a barrier to the proper development of SCNT embryos and, in particular, that expression of human histone H3K9me3 demethylase KDM4D/4A significantly improves the efficacy of both mouse and human SCNT [8,9]. Using transcriptomic analysis, they found that many genes are upregulated during the four- to eight-cell transition of SCNT embryos and that reactivation of some developmentally regulated pluripotency-associated genes requires the removal of the H3K9me3 epigenetic mark [1]. The authors then determined an optimal combination of the timing and concentration of H3K9me3 demethylase Kdm4d expression by mRNA injection and histone deacetylase inhibitor application using Trichostatin A. Using fetal skin fibroblasts from an aborted female cynomolgus monkey fetus as the source of nuclei, a total of 79 SCNT embryos between the two-cell to blastocyst stages were transferred to 21 cynomolgus female surrogates, leading to six pregnancies and two live births at full-term by caesarean section. These two baby monkeys, named ‘Zhongzhong’ and ‘Huahua’ remain healthy and growing (Fig. 1). The authors also performed similar experiments with adult cumulus cells of female monkeys as the donors. A total of 181 of SCNT embryos at similar stages were transferred to 42 surrogates, leading to 22 pregnancies and 2 live births by caesarean section, but both died within 30 h due to respiratory failure. It is not yet clear why adult somatic cells as a donor failed; potential reasons include epigenetic differences or telomere length between fetal and adult somatic cells. Importantly, genetic analysis of mitochondrial and nuclear DNAs confirmed the clonal origin of the four monkeys generated by the SCNT approach. Figure 1. View largeDownload slide An updated picture of ‘Zhongzhong’ (65 days old) and ‘Huahua’ (55 days old), two baby Macaque monkeys cloned by somatic cell nuclear transfer (Courtesy of Qiang Sun and Zhen Liu, Institute of Neuroscience, Chinese Academy of Sciences). Can you guess who is who? Figure 1. View largeDownload slide An updated picture of ‘Zhongzhong’ (65 days old) and ‘Huahua’ (55 days old), two baby Macaque monkeys cloned by somatic cell nuclear transfer (Courtesy of Qiang Sun and Zhen Liu, Institute of Neuroscience, Chinese Academy of Sciences). Can you guess who is who? This demonstration of the feasibility of reproductive cloning of macaque monkeys using somatic cell nuclei breaks a technical barrier and ushers in a new era of using non-human primates as experimental models. Because primate colonies normally require outbreeding to maintain genetic diversity for the health of the animals, the ability to generate a large number of genetically tractable and uniform non-human primates opens up countless opportunities. Reduced phenotypic variability will require much a smaller number of animals for a particular project. We can now generate a detailed reference database for these non-human primates on defined genetic backgrounds at the molecular, cellular, tissue and organ, and behavioral levels. Importantly, somatic cells, such as fetal skin fibroblasts, can be used for genome editing and screening, which is much more efficient than manipulating the embryos directly and avoids issues of mosaicism. Isogenic clones with modified genomes, such as knock-ins of reporters or human disease mutations, can be generated, phenotyped and compared to the reference database. These tools will not only be useful for increasing our understanding of basic biology and physiology, especially of primate-specific features, but will greatly facilitate biomedical discoveries and drug testing. For example, disease models can be used for discovery of early biomarkers for different disorders for diagnostics and for testing candidate therapeutic strategies. What are the challenges ahead? ‘Zhongzhong’ and ‘Huahua’ need to be closely monitored over the long-term for any signs of abnormality. Additional cloned monkeys with different genetic backgrounds are also needed for comparison. The current success provides a proof-of-principle case for the reprogramming competency of primate somatic nuclei, yet the efficacy of SCNT for non-human primates is still extremely low. With the conceptual barrier removed, it is likely that the technology will be rapidly improved. The dilemma is that the more efficient we become at SCNT cloning of monkeys, the closer it brings us to the ethical boundary of cloning humans. It is now time for an open discussion to establish guidelines, and perhaps international laws to prohibit even testing the possibility. Ethical guidelines are also needed for genome-editing in monkeys, given the possibility of knocking-in human-specific genes to achieve ‘humanized’ models, especially for the brain. In ‘Journey to the West’, a classic Chinese novel published in the 16th century, Monkey King, with a hair plucked from his body and a gentle blow, could produce unlimited copies of himself. While SCNT cloning of monkeys is not yet that simple in real life, this landmark study opens up a new chapter in the generation of non-human primate models for basic and translational research, and has the potential to accelerate the development of effective and safe new treatments for human diseases in the near future. REFERENCES 1. Liu Z , Cai Y , Wang Y et al. Cell 2018 ; 172 : 881 – 7.e7 . https://doi.org/10.1016/j.cell.2018.01.020 CrossRef Search ADS PubMed 2. Wilmut I , Bai Y , Taylor J . Phil Trans R Soc B 2015 ; 370 : 20140366 . https://doi.org/10.1098/rstb.2014.0366 CrossRef Search ADS PubMed 3. Gurdon JB , Elsdale TR , Fischberg M . Nature 1958 ; 182 : 64 – 5 . https://doi.org/10.1038/182064a0 CrossRef Search ADS PubMed 4. 童第周, 吴尚懃, 叶毓芬. 鱼类细胞核的移植. 科学通报 ; 1963 ; 14 : 60 – 1 . 5. Wilmut I , Schnieke AE , McWhir J et al. Nature 1997 ; 385 : 810 – 3 . https://doi.org/10.1038/385810a0 CrossRef Search ADS PubMed 6. Meng L , Ely JJ , Stouffer RL et al. Biol Reprod 1997 ; 57 : 454 – 9 . https://doi.org/10.1095/biolreprod57.2.454 CrossRef Search ADS PubMed 7. Chan AW , Dominko T , Luetjens CM et al. Science 2000 ; 287 : 317 – 9 . https://doi.org/10.1126/science.287.5451.317 CrossRef Search ADS PubMed 8. Antony J , Oback F , Chamley LW et al. Mol Cell Biol 2013 ; 33 : 974 – 83 . https://doi.org/10.1128/MCB.01014-12 CrossRef Search ADS PubMed 9. Chung YG , Matoba S , Liu Y et al. Cell Stem Cell 2015 ; 17 : 758 – 66 . https://doi.org/10.1016/j.stem.2015.10.001 CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

National Science ReviewOxford University Press

Published: Feb 15, 2018

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