• 한국임상수의학회지
• /
• 제36권5호
• /
• pp.253-258
• /
• 2019

The objective of this study was to analyse the effects of MS-275 (Class I and II histone deacetylase inhibitor) supplementation on the development of porcine in-vitro somatic nuclear transfer embryo production. During in-vitro development, early embryos were exposed to different concentrations of MS-275 (0, $5{\mu}M$, $10{\mu}M$, and $20{\mu}M$). In in-vitro culture supplemented group, the blastocyst development rate was significantly enhanced by $10{\mu}M$ concentration than other groups (24.0% vs. 19.3%, 21.8%, 11.5%; P < 0.05). Additionally, the 6 h supplementation group, significantly improved the blastocysts production than 24 h, 48 h and control groups (26.1% vs. 17.0%, 15.2%, 2.8%; P < 0.05). Following supplementation with optimal concentrations and time ($10{\mu}M$-6 h group), the blastocyst production was significantly higher than control (25.7% vs 15.8%; P < 0.05). The optimal concentrations of MS-275 significantly enhanced the percentages of ICM:TE than control (43.6% vs. 38.4%; P < 0.05) accompanied with significantly higher expression levels of reprogramming related genes (POU5F1, Naong, and SOX2). In conclusion, the optimal concentrations of $10{\mu}M$ MS-275 and 6 h supplementation during in-vitro culture can significantly improve the quality of porcine in-vitro somatic nuclear transfer embryos through histone acetylation and epigenetic modification. Increasing the efficiency of clonal animal production will greatly promote the development of animal disease models and xenotransplantation.

• BMB Reports
• /
• 제54권10호
• /
• pp.505-515
• /
• 2021

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) derived from blastocysts and human induced pluripotent stem cells (hiPSCs) generated from somatic cell reprogramming. Due to their self-renewal ability and pluripotent differentiation potential, hPSCs serve as an excellent experimental platform for human development, disease modeling, drug screening, and cell therapy. Traditionally, hPSCs were considered to form a homogenous population. However, recent advances in single cell technologies revealed a high degree of variability between individual cells within a hPSC population. Different types of heterogeneity can arise by genetic and epigenetic abnormalities associated with long-term in vitro culture and somatic cell reprogramming. These variations initially appear in a rare population of cells. However, some cancer-related variations can confer growth advantages to the affected cells and alter cellular phenotypes, which raises significant concerns in hPSC applications. In contrast, other types of heterogeneity are related to intrinsic features of hPSCs such as asynchronous cell cycle and spatial asymmetry in cell adhesion. A growing body of evidence suggests that hPSCs exploit the intrinsic heterogeneity to produce multiple lineages during differentiation. This idea offers a new concept of pluripotency with single cell heterogeneity as an integral element. Collectively, single cell heterogeneity is Janus-faced in hPSC function and application. Harmful heterogeneity has to be minimized by improving culture conditions and screening methods. However, other heterogeneity that is integral for pluripotency can be utilized to control hPSC proliferation and differentiation.

• Molecules and Cells
• /
• 제27권6호
• /
• pp.635-640
• /
• 2009

Testis-derived germline stem (GS) cells can undergo reprogramming to acquire multipotency when cultured under appropriate culture conditions. These multipotent GS (mGS) cells have been known to differ from GS cells in their DNA methylation pattern. In this study, we examined the DNA methylation status of the H19 imprinting control region (ICR) in multipotent adult germline stem (maGS) cells to elucidate how epigenetic imprints are altered by culture conditions. DNA methylation was analyzed by bisulfite sequencing PCR of established maGS cells cultured in the presence of glial cell line-derived neurotrophic factor (GDNF) alone or both GDNF and leukemia inhibitory factor (LIF). The results showed that the H19 ICR in maGS cells of both groups was hypermethylated and had an androgenetic pattern similar to that of GS cells. In line with these data, the relative abundance of the Igf2 mRNA transcript was two-fold higher and that of H19 was three fold lower than in control embryonic stem cells. The androgenetic DNA methylation pattern of the H19 ICR was maintained even after 54 passages. Furthermore, differentiating maGS cells from retinoic acid-treated embryoid bodies maintained the androgenetic imprinting pattern of the H19 ICR. Taken together these data suggest that our maGS cells are epigenetically stable for the H19 gene during in vitro modifications. Further studies on the epigenetic regulation and chromatin structure of maGS cells are therefore necessary before their full potential can be utilized in regenerative medicine.

• 한국가축번식학회지
• /
• 제27권4호
• /
• pp.309-315
• /
• 2003

DNA methylation at CpG sites, which is a epigenetic modification, is associated with gene expression without change of DNA sequences. During early mouse embryogenesis, dynamic changes of DNA methylation occur. In this study, DNA methylation patterns of porcine embryos produced in vivo and in vitro were examined at various developmental stages by the immunocytochemical staining method. Interestingly, active demethylation was not observed on the paternal pronucleus of porcine zygotes. However, differences were detected in the passive demethylation process between in vivo and in vitro embryos. There was no change in the DNA methylation state until the blastocyst stage of in vivo embryos, whereas partial demethylation was observed in several blastomeres from a 4 cell stage to a morula stage of in vitro embryos. The whole genome of inner cell mass (ICM) and trophectoderm (TE) cells in porcine blastocysts were evenly methylated without de novo methylation. Our findings demonstrate that genome-wide demethylation does not occur in pig embryos during preimplantation development unlike murine and bovine embryos. It indicates that the machinery regulating epigenetic reprogramming may be different between species.

• Asian-Australasian Journal of Animal Sciences
• /
• 제19권8호
• /
• pp.1090-1094
• /
• 2006

Histone acetylation modification is one key mechanism in the regulation of gene activation. In this study, we investigated the global levels of histone H4 acetylation of insulin like growth factor I (IGF1) and growth hormone (GH) genes in the lungs of two somatic cell cloned calves. Data showed the levels of histone H4 acetylation of IGF1 and GH genes vary widely within different gene regions, and, in almost all regions of the two genes, acetylation levels are lower in the aberrant clone than in the normal clone. Thus we suggest that inefficient epigenetic reprogramming in the clone may affect the balance between acetylation and deacetylation, which will affect normal growth and development. These findings will also have implications for improvement of cloning success rates.

• Molecules and Cells
• /
• 제20권3호
• /
• pp.423-428
• /
• 2005

Immediately after fertilization, a chromatin remodeling process in the oocyte cytoplasm extracts protamine molecules from the sperm-derived DNA and loads histones onto it. We examined how the histone H3-lysine 9 methylation system is established on the remodeled sperm chromatin in mice. We found that the paternal pronucleus was not stained for dimethylated H3-K9 (H3-$m_2K9$) during pronucleus development, while the maternal genome stained intensively. Such H3-$m_2K9$ asymmetry between the parental pronuclei was independent of $HP1{\beta}$ localization and, much like DNA methylation, was preserved to the two-cell stage when the nucleus appeared to be compartmentalized for H3-$m_2K9$. A conspicuous increase in H3-$m_2K9$ level was observed at the four-cell stage, and then the level was maintained without a visible change up to the blastocyst stage. The behavior of H3-$m_2K9$ was very similar, but not identical, to that of 5-methylcytosine during preimplantation development, suggesting that there is some connection between methylation of histone and of DNA in early mouse development.

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

• 한국가금학회지
• /
• 제41권3호
• /
• pp.217-225
• /
• 2014

텔로미어는 진핵 세포의 염색체 말단으로, 직렬 반복 DNA 염기 서열과 shelterin 단백질 복합체로 구성되어 있다. 텔로미어의 기능은 염색체를 보호하는 것으로 체세포의 텔로미어 길이는 세포 분열시 DNA 복제 결실로 인해 연령이 증가함에 따라 감소하는 경향이 있다. 그러나 유전적, 후생유전학적 및 환경적 수준에서 여러 가지 요인이 텔로미어 길이에 영향을 미친다. 따라서 본 연구에서는 닭의 텔로미어 길이의 유전력을 추정하고, 이들의 유전전이 양상을 살펴보고자 하였다. 텔로미어 길이는 백혈구를 이용하여 양적 형광접합보인법(Q-FISH)과 양적 중합효소 연쇄반응법(qRT-PCR)으로 분석하였다. 분석 결과, 텔로미어 길이의 유전력은 자손과 부모 회귀 분석에 의해 출생 시 0.9로 추정되었고, 10 주령 및 30주령 때 부 분산 분석에 의해 0.03과 0.04로 추정되었다. 부와 자손 간(r=0.348) 및 모와 자손 간(r=0.380) 텔로미어 길이는 모두 유의한 정의 상관 관계를 보였다. 따라서 닭 텔로미어의 유전 전이 양상은 부모 양쪽 모두로부터 비슷하게 자식에 영향을 미치는 것으로 판단된다. 더불어 암수 자손에 미치는 영향 또한 유사한 것으로 나타났다. 이러한 결과는 부모의 텔로미어 길이의 각인이 성염색체의 유전자가 아닌 상염색체의 유전자에 의해 조절되는 것을 의미한다. 또한, 산모 연령에 따른 출생 자손의 텔로미어 길이는 차이가 없는 것으로 나타났다. 따라서 모체의 연령이 출생 자손의 텔로미어 길이에 영향을 미치지 않는데, 이는 수정란의 초기 배아 단계에서 세포적 reprogramming이 이루어지기 때문으로 사료된다.

• 한국발생생물학회:학술대회논문집
• /
• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
• /
• pp.81-81
• /
• 2003

DNA methylation is a covalent modification of DNA that can modulate gene expression and is now recognized as a major component of the epigenome. During evolution, the dinucleotide CpG has been progressively eliminated from the genome of higher eukaryotes and is present at only 5% to 10% of its predicted frequency. Approxymately 80% of the remaining CpG sites contain methylated cytosines in most vertebrates and they are distributed in a pattern that is unique in each tissue and is inversely correlated with gene expression. The pattern of methylation is faithfully maintained during cell division by the enzyme Dnmt1, the maintenance DNA methyltransferase, which catalyzes the transfer of a methyl group from S-adenosyl-methionine to the 5'-position of the cytosine ring. We have been identified bovine Dnmt1 cDNA full-length recently (AY173048) Little is known on the functions of Dnmt1 in bovine preimplantation embryos. Thus, we analyzed the specific pattern of Dnmt1 in in vitro derived/nuclear transfer bovine and in vivo derived mouse embryos to monitor the epigenetic reprogramming process. We investigated these process by using indirect immunofluresence with an antibody to Dnmt1. According to other studies, Dnmt1 accumulates in nuclei of early growing oocytes but is sequestered in the cytoplasm of mature oocytes. In 2-cell and 4-cell embryos, Dnmt1 is cytoplasmic, but at the 8-cell stage, it is present only in the nucleus. By the blastocyst stage, Dnmt1o is again found only in the cytoplasm. Thus, nuclear localization of Dnmt1o in preimplantation embryos is limited to the 8-cell stages After implantation, Dnmt1 is localized in the nucleus in mouse. However, we have found different patterns of Dnmt1 nuclear localization. Though we used the common antibody, immune-localization data revealed that Dnmt1 antibody have been detected at the nucleus in 1-cell to blastocyst embryos. Therefore, maybe we think that the functions of Dnmt1 between bovine and mice are different. In order to Identify the mechanisms that regulate DNA methylation in bovine preimplantation embryo, we have plans on using bovine oocyte and somatic specific Dnmt1 antibodies.