• Korean Journal of Veterinary Research
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• v.55 no.3
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• pp.155-161
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• 2015

The number of wild animal species is gradually decreasing due to poaching, hunting and habitat loss. While several endangered animal species have been successfully preserved at the zoo, assisted reproductive technology (ART) must be applied to restore wild animals. In the case of critically endangered animals, somatic cell cloning is considered the most appropriate method of ART. Somatic cell cloning can be beneficial for the reproduction of endangered species with limited female populations. However, gene and cell banks, and understanding of reproductive physiology and optimization of ART for wild animals are urgently required for further activation of artificial reproduction of endangered species, which enlarges its application and maintains biodiversity. Care should also be taken to consider ethical and legal issues associated with somatic cell cloning for conservation of endangered animals.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.7-8
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• 2003

Since it was first reported in 1997, somatic cell cloning has been demonstrated in several other mammalian species. On the mouse, it can be cloned from embryonic stem (ES) cells, fetus-derived cells, and adult-derived cells, both male and female. While cloning efficiencies range from 0 to 20%, rates of just 1-2% are typical (i.e. one or two live offspring per one hundred initial embryos). Recently, abnormalities in mice cloned from somatic cells have been reported, such as abnormal gene expression in embryo (Boiani et al., 2001, Bortvin et al., 2003), abnormal placenta (Wakayama and Yanagimachi 1999), obesity (Tamashiro et ai, 2000, 2002) or early death (Ogonuki et al., 2002). Such abnormalities notwithstanding, success in generating cloned offspring has opened new avenues of investigation and provides a valuable tool that basic research scientists have employed to study complex processes such as genomic reprogramming, imprinting and embryonic development. On the other hand, mouse ES cell lines can also be generated from adult somatic cells via nuclear transfer. These 'ntES cells' are capable of differentiation into an extensive variety of cell types in vitro, as well assperm and oocytes in vivo. Interestingly, the establish rate of ntES cell line from cloned blastocyst is much higher than the success rate of cloned mouse. It is also possible to make cloned mice from ntES cell nuclei as donor, but this serial nuclear transfer method could not improved the cloning efficiency. Might be ntES cell has both character between ES cell and somatic cell. A number of potential agricultural and clinical applications are also are being explored, including the reproductive cloning of farm animals and therapeutic cloning for human cell, tissue, and organ replacement. This talk seeks to describe both the relationship between nucleus donor cell type and cloning success rate, and methods for establishing ntES cell lines. (중략)

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.393-398
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• 1999

Somatic cell nuclear transfer is an efficient technique for the multiplication of elite livestock, engineering of transgenic animals, cell therapy and xenotransplantation, and analyzing the interactions between nucleus and cytoplasm, for various agricultural, biomedical and research purposes. Since the first somatic cell clone lamb was born, tremendous progress has been made toward developing technology for animal cloning. Viable farm animals and mice have now been produced by nuclear transfer using various fetal and adult somatic cells as nuclei donors. Transgenic clones were also produced from nuclear transfer of transfected somatic cells. In the future, somatic cell nuclear transfer will provide more numerous opportunities, both in basic and appled research as well as immediate uses in the generations of superior clone and transgenic animals. However, further technology refinement and improved understanding of the process are essential for commercial and basic research applications.

• Journal of Embryo Transfer
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• v.23 no.2
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• pp.67-76
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• 2008

Since the birth of Dolly using fully differentiated somatic cells as a nuclear donor, viable clones were generated successfully in many mammalian species. These achievements in animal cloning demonstrate developmental potential of terminally differentiated somatic cells. At the same time, the somatic cell nuclear transfer (SCNT) technique provides the opportunities to study basic and applied biosciences. However, the efficiency generating viable offsprings by SCNT remains extremely low. There are several explanations why cloned embryos cannot fully develop into viable animals and what factors affect developmental potency of reconstructed embryos by the SCNT technique. The most critical and persuasive explanation for inefficiency in SCNT cloning is incomplete genomic reprogramming, such as DNA methylation and histone modification. Numerous studies on genomic reprogramming demonstrated that incorrect DNA methylation and aberrant epigenetic reprogramming are considerably correlated with abnormal development of SCNT cloned embryos even though its mechanism is not fully understood. The SCNT technique is useful in cloning farm animals because pluripotent stem cells are not established in farm animal species. Therapeutic cloning combined with genetic manipulation will help to control various human diseases. Also, the SCNT technique provides a chance to overcome excessive demand for the organs by production of transgenic animals as xenotransplantation resources. Here, we describe the factors affecting the efficiency of generating cloned farm animals by the SCNT technique and discuss future directions of animal cloning by SCNT to improve the cloning efficiency.

• Journal of Animal Science and Technology
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• v.63 no.2
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• pp.281-294
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• 2021

Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.43-55
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• 2002

Cloning by nuclear transfer in mammals using somatic cells has enormous potential applications. However, somatic cloning has been inefficient in all species in which NT is successful. High abortion and fetal death rates have been observed. These developmental defects have been attributed to incomplete nuclear reprogramming by the somatic cloning process. In this review, we will discuss studies conducted in our labs to understand the nuclear reprogramming process.

• 대한생식의학회:학술대회논문집
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• 2000.02a
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• pp.229-234
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• 2000

Transgenic animal technology has provided new opportunities in many aspects of biotechnology and medicine during two decades. Several gene delivery systems including pronuclear injection, retroviral vectors, sperm vectors, and somatic cell cloning have been tried to generate new functional animals. In the future somatic cell cloning technology will be a major method in the transgenic animal production. Many factors enhancing overall transgenic efficiency should be overcome to facilitate the industrial applications of transgenic technology. Transgenic animal technology has settled down in some areas of the medicine, especially the mass production of pharmaceutical proteins and xenotransplantation. Thus, animal biotechnology will contribute to welfare of human being.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2001.10a
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• pp.37-43
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• 2001

1. About fifty thousand of cattle embryos were transferred and 16000 ET-calves were born in 1999. Eighty percents of embryos were collected from Japanese Black beef donors and transferred to dairy Holstein heifers and cows. Since 1985, we have achieved in bovine in vitro fertilization using immature oocytes collected from ovaries of slaughterhouse. Now over 8000 embryos fertilized by Japanese Black bull, as Kitaguni 7~8 or Mitsufuku, famousbulls as high marbling score of progeny tests were sold to dairy farmers and transferred to their dairy cattle every year. 2. Embryo splitting for identical twins is demonstrated an useful tool to supply a bull for semen collection and a steer for beef performance test. According to the data of Dr. Hashiyada(2001), 296 pairs of split-half embryos were transferred to recipients and 98 gave births of 112 calves (23 pairs of identical twins and 66 singletons). 3. A blastomere-nuclear-transferred cloned calf was born in 1990 by a joint research with Drs. Tsunoda, National Institute of Animal Industry (NIAI) and Ushijima, Chiba Prefectural Farm Animal Center. The fruits of this technology were applied to the production of a calf from a cell of long-term-cultured inner cell mass (1988, Itoh et al, ZEN-NOH Central Research Institute for Feed and Livestock) and a cloned calf from three-successive-cloning (1997, Tsunoda et al.). According to the survey of MAFF of Japan, over 500 calves were born until this year and a glaf of them were already brought to the market for beef. 4. After the report of "Dolly", in February 1997, the first somatic cell clone female calves were born in July 1998 as the fruits of the joint research organized by Dr. Tsunoda in Kinki University (Kato et al, 2000). The male calves were born in August and September 1998 by the collaboration with NIAI and Kagoshima Prefecture. Then 244 calves, four pigs and a kid of goat were now born in 36 institutes of Japan. 5. Somatic cell cloning in farm animal production will bring us as effective reproductive method of elite-dairy- cows, super-cows and excellent bulls. The effect of making copy farm animal is also related to the reservation of genetic resources and re-creation of a male bull from a castrated steer of excellent marbling beef. Cloning of genetically modified animals is most promising to making pig organs transplant to people and providing protein drugs in milk of pig, goat and cattle. 6. Farm animal cloning is one of the most dreamful technologies of 21th century. It is necessary to develop this technology more efficient and stable as realistic technology of the farm animal production. We are making researches related to the best condition of donor cells for high productivity of cloning, genetic analysis of cloned animals, growth and performance abilities of clone cattle and pathological and genetical analysis of high rates of abortion and stillbirth of clone calves (about 30% of periparutum mortality). 7. It is requested in the report of Ministry of Health, labor and Welfare to make clear that carbon-copy cattle(somatic cell clone cattle) are safe and heathy for a commercial market since the somatic cell cloning is a completely new technology. Fattened beef steers (well-proved normal growth) and milking cows(shown a good fertility) are now provided for the assessment of food safety.

• Proceedings of the Zoological Society Korea Conference
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• 1999.10b
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• pp.6-7
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• 1999

No Abstract, See Full Text

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.3
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• pp.215-222
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• 2020

Mesenchymal stem cells (MSCs) have been widely used as donor cells for somatic cell nuclear transfer (SCNT) to increase the efficiency of embryo cloning. Since replicative senescence reduces the efficiency of embryo cloning in MSCs during in vitro expansion, transfection of telomerase reverse transcriptase (TERT) into MSCs has been used to suppress the replicative senescence. Here, TERT-transfected MSCs in comparison with early passage MSCs (eMSCs) and sham-transfected MSCs (sMSCs) were used to evaluate the effects of embryo cloning with SCNT in a porcine model. Cloned embryos from tMSC, eMSC, and sMSC groups were indistinguishable in their fusion rate, cleavage rate, total cell number, and gene expression levels of OCT4, SOX2 and NANOG during the blastocyst stage. The blastocyst formation rates of tMSC and sMSC groups were comparable but significantly lower than that of the eMSC group (p < 0.05). In contrast, tMSC and eMSC groups demonstrated significantly reduced apoptotic incidence (p < 0.05), and decreased BAX but increased BCL2 expression in the blastocyst stage compared to the sMSC group (p < 0.05). Therefore, MSCs transfected with telomerase reverse transcriptase do not affect the overall development of the cloned embryos in porcine SCNT, but enables to maintain embryo quality, similar to apoptotic events in SCNT embryos typically achieved by an early passage MSC. This finding offers a bioengineering strategy in improving the porcine cloned embryo quality.