• BMB Reports
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• 제44권8호
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• pp.535-540
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• 2011

Reprogramming errors, which appear frequently in cloned animals, are reflected by aberrant gene expression. We previously reported the aberrant expression of TIMP-2 and PBEF in cloned placenta and differential expression of PBEF genes during pregnancy. To examine the epigenetic modifications that regulate dynamic gene expression in developing placentae, we herein analyzed the mRNA and protein expression levels of PBEF and TIMP-2 in the placentae of normal mice during pregnancy and then examined potential correlations with epigenetic modifications. DNA methylation pattern analysis revealed no difference, but ChIP assays using antibodies against H3-K9/K14 and H4-K5 histone acetylation revealed that the H3-K9/K14 acetylation levels, but not the H4-K5 acetylation levels, of the TIMP-2 and PBEF loci were significantly correlated with their gene expression levels during placentation in normal mice. These results suggest that epigenetic changes may regulate gene expression level in the developing placentae of normal mice and that inappropriate epigenetic reprogramming might be one cause of the abnormal placentae seen in cloned animals.

• Reproductive and Developmental Biology
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• 제33권4호
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• pp.195-202
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• 2009

Abnormal development and fetal loss during the post-implantation period are key concerns in the production of cloned animals by somatic cell nuclear transfer (SCNT). We hypothesized that the problems in cloned porcine offspring derived from SCNT are related to interactions between the conceptus and the endometrial environment. In the present study, we investigated expression patterns in the formation of placenta-related genes (Cdx2 and GATA6) in whole in vivo normal porcine embryos (from single cell to blastocyst) and each tissue of a normal fetus at Days 25, 35 and 55 by quantitative mRNA expression analysis using real-time PCR. The expression of Cdx2 and GATA6 mRNA increased to around the blastocyst stage. These genes were gradually decreased from the peri-implantation to post-implantation stage. Moreover, we examined the expression patterns of Cdx2 and GATA6 in Day 35 normal and SCNT cloned fetuses by the same methods. And, the level of Cdx2 and GATA6 gene expression in the extraembryonic tissue of SCNT was significantly higher than that of control tissues. From the present results, it can be postulated that the aberrant expression of Cdx2 and GATA6 genes in the endometrial and extraembryonic tissues at pre- and peri-implantation stages may be closely related to the lower efficiency of animal cloning.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.82-82
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• 2003

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.197-197
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• 2004

The purpose of this experiment was to investigate the protein profiles in the placenta of Korean native cows(KNC) transferred cloned embryos and KNC artificially inseminated placental tissues were collected from the cows after cesarean section around parturition, and placental proteins were analyzed. Using two dimensional polyacrylamide gel eletrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. (omitted)

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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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)

• 한국발생생물학회지:발생과생식
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• 제4권1호
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• pp.87-93
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• 2000

eCG는LH, FSH및 TSH와 같이 당단백질 호르몬에 속하고, 당쇄가 많이 첨가된 $\alpha$와 $\beta$-subunits의 비공유결합으로 구성되어 있고, 말에서 보다 다른 동물에서 FSH와 LH의 이중 생리활성을 나타내는 아주 특이한 성선 자극 호르몬이다. eCG는 임신 40~130일 사이에 말의 자궁내막배의 영양막세포에서 합성ㆍ분비된다. 따라서 본 연구에서는 eCG, eLH 및 eFSH의 각각 subunits mRNA발현을 태반과 뇌하수체에서 분석하였다. mRNA의 추출은 임신 70일의 태반과 27개월된 숫컷말의 뇌하수체에서 분리하였다. 말 태반을 이용한eCG mRNA발현의 Northern blotting분석결과 $\beta$ subunit가 $\alpha$ subunit보다 아주 많이 발현되었으며, 또한 뇌하수체에서 $\alpha$-, LH $\beta$-, FSH $\beta$-subunit의 분석결과 $\alpha$ subunit는 약 0.8 kb, FSH $\beta$ subunit는 1.8 kb의 크기로 발현되었는데, 이러한 FSH $\beta$ subunit는 cloning되어진 cDNA의 크기와 일치한다. 뇌하수체 전엽에서는 $\alpha$ subunit가 LH $\beta$ subunit와 FSH $\beta$ subunit보다 현저히 많이 발현된다는 사실이 밝혀졌다. 따라서, 태반과 뇌하수체에서 발현되는 각각 subunit의 mRNA는 독립적으로 조절되어 결과적으로 발현량에 차이가 나타난다고 시사되어진다.

• 한국수정란이식학회지
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• 제29권2호
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• pp.111-117
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• 2014

This study was conducted to optimize the efficiency of cloning and to produce cloned mice. The majority of cloned mammals derived by nuclear transfer (NT) die during gestation and have enlarged and dysfunctional placentas. In this study, the optimized conditions were established to produce clone mice. The parthenogenetic oocytes were activated after 6 h regardless of cytochalasin B (CB) concentration. CB treatment ($2{\mu}g/ml$) was found second polar body. Lower concentration of CB was decreased the activation rate, but the second polar body was the best highly increased during 6 h incubation. The small fragments were exhibited in the $5{\mu}g/ml$ treatment of CB, but it was not found in lower concentration groups (> $2.5{\mu}g/ml$). To examine effects of $SrCl_2$ on the adult cumulus cells, somatic cell NT oocytes were exposed during 0.5, 1 and 6 hrs. The second polar body was significantly greater in 0.5 h exposure group (6.6%) than 1, 6 hrs. Developmental rate from 2-cell to 4-cell was the lowest in 7.5 mM Strontium chloride ($SrCl_2$) groups (84.1% and 64.3%) than 5, 10 m $MSrCl_2$. The implantation rate was not significantly difference among 5, 7.5 and 10 m $MSrCl_2$ group. Three live fetuses were produced by SCNT. SCNT placentas were remarkably heavier than IVF group (8 fetuses) (0.34, 0.34, 0.33 vs 0.14 g) compared with the placenta weight of IVF and SCNT clones.

• Reproductive and Developmental Biology
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• 제29권2호
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• pp.83-91
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• 2005

Somatic cell nuclear transfer in cattle has limited efficiency in terms of production of live offspring due to high incidence of fetal failure after embryo transfer to recipients. Such low efficiency of cloning could possibly arise from abnormal and poorly developed placenta. In the present study the placental proteome in late pregnancy established from in vitro fertilization (IVF) and nuclear transfer (NT) was analysed. Proteome alternation was tested using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF). Comparing placenta from NT embryos to those from IVF counterparts, significant changes in expression level were found in 18 proteins. Of these proteins 12 were not expressed in NT placenta but expressed in IVF counterpart, whereas the expression of the other 6 proteins was limited only in NT placenta. Among these proteins, cytokeratin 8 and vimentin are considered to be involved in regulation of post-implantation development. In particular, cytokeratin 8 and vimentin may be used as makers for placental development during pregnancy because their expression levels changed considerably in NT placental tissue compared with its IVF counterpart. Data from 2-DE suggest that protein expression was disorientated in late pregnancy from NT, but this distortion was eliminated with progression of pregnancy. These findings demonstrate abnormal placental development during late pregnancy from NT and suggest that alterations of specific placental protein expression may be involved in abnormal function of placenta.

• 한국산학기술학회논문지
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• 제20권8호
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• pp.477-484
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• 2019

포유류에 존재하는 Collectin-Placenta 1 (CL-P1)을 포함한 여러 종류의 scavenger 수용체는 주로 내피 세포, 대식 세포 및 평활근 세포 표면에 발현되는 분자이다. 이들 분자는 산화 된 저밀도 지질 단백질 (oxLDL)에 결합하여 처리 할 수 있는 세포 표면 당 단백질이다. 이들 분자 중 케르세틴이 CL-P1 활성화에 어떤 영향을 미치는가를 확인하였다. 케르세틴은 산화 반응을 담당하는 자유 라디칼의 제거제 역할을 하여 산화를 중지시키는 항산화제로 알려져 있다. 본 논문에서는 마우스 CL-P1 유전자 promoter 부분의 전사 시작 점부터 -500 번째 염기까지의 단편을 DNA 중합효소를 이용하여 클로닝 하였다. 그 후에 대식세포 계열인 RAW264.7 및 섬유아세포계열의 NIH3T3 세포에 도입하여 케르세틴이 CL-P1 유전자 발현에 어떠한 영향을 미치는지에 대한 연구를 하였다. 이 부위에는 세포주기 조절 인자인 E2F 결합부위를 비롯해서 여러 종류의 전사 인자가 결합하는 염기서열이 다수 위치하고 있다. 이러한 500염기 단편을 pGL4.10 기본 벡터 및 프로모터에 연결시킨 후 세포에 도입시켰다. 그리고 배양 중에 케르세틴을 처리하여 유전자 발현양을 형광 색소 발현기법으로 측정하였다. 그 결과 유전자 발현이 시작되는 앞쪽 부분의 -250에서 -350사이의 염기들이 CL-P1 단백질을 만드는데 중요하다는 것을 확인하였다. 그 중에서도 E2F결합 부위가 결정적인 것 이라는 것을 DNA 돌연변이 실험을 통해 확인 하였다. 또한 부착 세포인 RAW264.7 배양액에 케르세틴을 첨가 한 결과, 배양용기 표면에서 탈락하는 현상을 확인하였다. 즉 발현된 CL-P1단백질이 케르세틴에 의해 세표 표면의 부착 분자에도 영향을 주는 것을 확인하였다.

• 한국수정란이식학회지
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• 제26권1호
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• pp.79-84
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• 2011

This study was performed to identify the differentially methylated region (DMR) and to examine the mRNA expression of the imprinted H19 gene in day 35 of SCNT pig fetuses. The fetus and placenta at day 35 of gestation fetuses after natural mating (Control) or of cloned pig by somatic cell nuclear transfer (SCNT) were isolated from a uterus. To investigate the mRNA expression and methylation patterns of H19 gene, tissues from fetal liver and placenta including endometrial and extraembryonic tissues were collected. The mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. Bisulfite analyses demonstrated that the differentially methylated region (DMR) was located between -1694 bp to -1338 bp upstream from translation start site of the H19 gene. H19 DMR (-1694 bp to -1338 bp) exhibits a normal mono allelic methylation pattern, and heavily methylated in sperm, but not in oocyte. In contrast to these finding, the analysis of the endometrium and/or extraembryonic tissues from SCNT embryos revealed a complex methylation pattern. The DNA methylation status of DMR Region In porcine H19 gene upstream was hypo methylated in SCNT tissues but hypermethylated in control tissues. Furthermore, the mRNA expression of H19 gene in liver, endometrium, and extraembryonic tissues was significantly higher in SCNT than those of control (p<0.05). These results suggest that the aberrant mRNA expression and the abnormal methylation pattern of imprinted H19 gene might be closely related to the inadequate fetal development of a cloned fetus, contributing to the low efficiency of genomic reprogramming.