• 한국수정란이식학회지
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• 제23권2호
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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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• 제63권2호
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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.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.29-48
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• 2003

It is remarkable that nuclear transfer using differentiated donor cells can produce physiologically normal cloned animals, but the process is inefficient and highly prone to epigenetic errors. Aberrant patterns of gene expression in clones contribute to the cumulative losses and abnormal phenotypes observed throughout development. Any long lasting effects from cloning, as revealed in some mouse studies, need to be comprehensively evaluated in cloned livestock. These issues raise animal welfare concerns that currently limit the acceptability and applicability of the technology. It is expected that improved reprogramming of the donor genome will increase cloning efficiencies realising a wide range of new agricultural and medical opportunities. Efficient cloning potentially enables rapid dissemination of elite genotypes from nucleus herds to commercial producers. Initial commercialization will, however, focus on producing small numbers of high value animals for natural breeding especially clones of progeny-tested sires, The continual advances in animal genomics towards the identification of genes that influence livestock production traits and human health increase the ability to genetically modify animals to enhance agricultural efficiency and produce superior quality food and biomedical products for niche markets. The potential opportunities in animal agriculture are more challenging than those in biomedicine as they require greater biological efficiency at reduced cost to be economically viable and because of the more difficult consumer acceptance issues. Nevertheless, cloning and transgenesis are being used together to increase the genetic merit of livestock; however, the integration of this technology into farming systems remains some distance in the future.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.29-48
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• 2003

It is remarkable that nuclear transfer using differentiated donor cells can produce physiologically normal cloned animals, but the process is inefficient and highly prone to epigenetic errors. Aberrant patterns of gene expression in clones contribute to the cumulative losses and abnormal phenotypes observed throughout development. Any long lasting effects from cloning, as revealed in some mouse studies, need to be comprehensively evaluated in cloned livestock. These issues raise animal welfare concerns that currently limit the acceptability and applicability of the technology. It is expected that improved reprogramming of the donor genome will increase cloning efficiencies realising a wide range of new agricultural and medical opportunities. Efficient cloning potentially enables rapid dissemination of elite genotypes from nucleus herds to commercial producers. Initial commercialisation will, however, focus on producing small numbers of high value animals for natural breeding especially clones of progeny-tested sires. The continual advances in animal genomics towards the identification of genes that influence livestock production traits and human health increase the ability to genetically modify animals to enhance agricultural efficiency and produce superior quality food and biomedical products for niche markets. The potential opportunities inanimal agriculture are more challenging than those in biomedicine as they require greater biological efficiency at reduced cost to be economically viable and because of the more difficult consumer acceptance issues. Nevertheless, cloning and transgenesis are being used together to increase the genetic merit of livestock; however, the integration of this technology into farming systems remains some distance in the future.

• 약학회지
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• 제31권2호
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• pp.116-125
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• 1987

It is still difficult and time consuming to obtain cDNA sequences that contain the entire nucleotide sequence of the corresponding mRNA. A rapid and high efficient cloning method to obtain full-length cDNA segments is thus developed. The cloning procedure described here consists of the construction of oligo(dT)-tailed vector primer using pWR34 plasmid, polyadenylation of mRNA-cDNA heteroduplex using terminal deoxytransferase, and replacement of MRNA strand with DNA by RNase H and DNA polymerase I. The restriction endonuclease analysis shows that the size of inserted-cDNA is in the range of 1.5~4.0 kb long suggesting that most of cloned cDNA are full-length or nearly full-length cDNA. The plasmid-DNA recombinants obtained were 4$\times$$10^5$~$10^{6}$ per $\mu\textrm{g}$ of rat liver poly (A$^+$)mRNA, which is 4 to 10 fold higher cloning efficiency in comparison to the presently used methods for full-length cDNA cloning. The results indicate that the described cloning system is much simpler, less time consuming, and very efficient cloning method to construct a cDNA library.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 1998년도 춘계학술대회 및 워크숍
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• pp.12-28
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• 1998

The technology of creating transgenic animals has a potential value in improving productivity and disease resistance of animals, gene therapy, drug pharming and production of model animals for certain diseases. Up to date, fairly low success rate of production of transgenic animals and a pronounced variability with respect to the expression of transgenes have been much observed. The mechanisms how to integrate the injected genes with a certain part of the genomes are unknown yet. Many techniques in gene transfer, beside microinjection, have been introduced and explored thus to improve the production efficiency of transgenic animals. In this article, the methods and efficiency of gene-transfer techniques, the detection and preselection of transgenes in embryos by PCR- and GFP-screenings and cloning of preselected transgenic embryos by nuclear transplantation are described and discussed. Some experimental results showed that the early screening and selection of integration of the injected gene with embryonic genome by polymerase chain reaction(PCR) and green fluorecence protein(GFP) were promising methods. Further, the application of nuclear transplantation technology to cloning and multiplication of the positively integrated genes in the cleaving embryos and embryonic cells will be beneficially used for the mass production of transgenic embryos and consequently improving the production efficiency in transgenic animals.

• 미생물학회지
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• 제39권3호
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• pp.128-134
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• 2003

Transformation-associated recombination (TAR) 클로닝법은 목적 유전자를 포함한 게놈 DNA와 그 유전자의 5' 또는 3' 말단 서열을 포함하고 있는 선형의 TAR vector를 동시에 출아효모의 spheroplast내로 co-penetration 시켜 상동부위에서 일어나는 재조합에 의해 환형의 Yeast Artification Chromosome(YAC)으로 분리되는 방법이다. 일반적으로 TAR 클로닝법에 의한 목적의 single-copy 유전자 분리 빈도는 전체 형질전환체의 0.01~1% 정도이다. 이러한 TAR 클로닝법을 개선하기 위하여 Tg.AC transgenic mouse를 모델계로 사용하여 유전자 분리에 대한 target hook 내의 GC content 가 미치는 영향을 조사하였다. 이러한 목적으로 한쪽에는 다양한 GC content(18~45%)를 지닌 transgene 특이적 hook을 포함하고 다른 한쪽은 B1 반복서열을 가지는 radial TAR vector를 사용하여 transgene 분리 빈도를 측정하였다. 그 결과 target hook의 GC content는 23% 이하의 경우, ~40%인 경우에 비해 두 배 정도 클로닝 빈도가 감소하였다. 따라서 TAR vector를 제작할 때, 유전자 분리에 이용되는 target hook의 GC content는 약 40% 일때 가장 적정한 것으로 나타났다. 또한 높은 target hook 내의 GC content(65%)위치분포에 의한 차이는 클로닝 빈도에 큰 영향을 미치지 않는 것으로 나타났다.

• 한국임상수의학회지
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• 제16권1호
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• pp.163-176
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• 1999

The nuclear transplantation technique is known as the most potential and efficient method for producing large numbers of genetically identical animals from a single embryo and somatic cells. After Dolly was introduced in 1997, many scientists were amazed. A possibility came to a reality that live offspring could be produced with differentiated somatic cells from an adult animal. On the other side, many in the press and the sensationalists focused on the socially, ethically and scientifically unacceptable sides of the technology. In this article, the history, current status and prospects of the technological development of nuclear transplantation in mammals and its application to the production of cloned animals are described. For the efficient and successful production of cloned embryos by nuclear transplantation, the right selection, preactivation and micromanipulation of oocytes as capacious recipient cytoplasm, the adequate and benefitial preparation of multiple totipotent embryonic and somatic cells as donor nuclei, fusion of them and in vitro production of cloned embryos are very critical. Recently the overall efficiency of production of cloned embryos and offspring in livestock has been much improved. Cloning will also be a more efficient, faster and useful way of creating transgenic fetuses for gene therapies, gene pharming, organs for xenotransplantation by preselection and mass production of transgenic embryos and consequently improving the production efficiency in transgenic animals. Further technical development of nuclear transplantation will enable large-scale production of cloned livestock and in near future the commercial cloning of animals will become a reality.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 제1차 연수강좌
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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.

• Animal Bioscience
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• 제35권2호
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• pp.177-183
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• 2022

Objective: The present study evaluated the efficiency of embryo development and pregnancy of somatic cell nuclear transfer (SCNT) embryos using different source-matured oocytes in Camelus dromedarius. Methods: Camelus dromedarius embryos were produced by SCNT using in vivo- and in vitro- matured oocytes. In vitro embryo developmental capacity of reconstructed embryos was evaluated. To confirm the efficiency of pregnancy and live birth rates, a total of 72 blastocysts using in vitro- matured oocytes transferred into 45 surrogates and 95 blastocysts using in vivo- matured oocytes were transferred into 62 surrogates by transvaginal method. Results: The collected oocytes derived from ovum pick up showed higher maturation potential into metaphase II oocytes than oocytes from the slaughterhouse. The competence of cleavage, and blastocyst were also significantly higher in in vivo- matured oocytes than in vitro- matured oocytes. After embryo transfer, 11 pregnant and 10 live births were confirmed in in vivo- matured oocytes group, and 2 pregnant and 1 live birth were confirmed in in vitro- matured oocytes group. Furthermore, blastocysts produced by in vivo-matured oocytes resulted in significantly higher early pregnancy and live birth rates than in vitro-matured oocytes. Conclusion: In this study, SCNT embryos using in vivo- and in vitro-matured camel oocytes were successfully developed, and pregnancy was established in recipient camels. We also confirmed that in vivo-matured oocytes improved the development of embryos and the pregnancy capacity using the blastocyst embryo transfer method.