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장석민,강재구,최기명,김홍래,진동일 충남대학교 농업과학연구소 2007 농업과학연구 Vol.34 No.1
This study was conducted to evaluate the response of CIDR-S on estrus synchronization of dairy goats and the effect of artificial insemination on conception rate. Estrus synchronization of sexually matured Saanen goats was induced with insertion of CIDR-S into vagina for 15 days and injection of 500 IU PMSG before removal of CIDR-S. Artificial insemination was conducted using liquid or frozen semen by intra-cervical insemination with synchronized does. Estrus synchronization was 90% using CIDR-S insertion for 15 days. Conception rate of inseminated does was 20% - 25%, while that of natural mated does was 100%. These results suggested that synchronization of dairy goats can be successfully induced and artificial insemination method has to be improved for the practical use in dairy goats.
Kim, Baek-Chul,Kim, Hong-Rye,Kim, Myung-Yoon,Park, Chang-Sik,Jin, Dong-Il The Korean Society of Animal Reproduction 2009 Reproductive & developmental biology Vol.33 No.2
Animals produced by somatic cell nuclear transfer (SCNT) using genetically modified cells are almost always transgenic, implying that this method is more efficient than the traditional pronuclear microinjection method. Most somatic cells for SCNT in animals are fetus-derived primary cells and successful gene integration in somatic cells will depend on transfection condition. The objective of this study is to evaluate the efficiency of electroporation (Microporator) and liposome reagents (F-6, F-HD, W-EX, W-Q, W-M) for tissue-type plasminogen activator (tPA) gene transfection and to estimate the overall efficiency of transfection of Korean native pig fetal fibroblast cells (KNPFF). Electroporation showed significantly higher transfection efficiency than liposome reagents with regard to the transfection of in vitro cultures in the early stages of development (41.7% with Microporator vs. 18.3% with F-6, 20.0% with F-HD 18.5% with W-EX, 5.0% with W-M and 6.3% W-Q,). Colonies identified as tPA-positives were treated once more with G418 for 10 to 14 days and growing colonies were selected again. When the cells of newly selected colonies were subjected to single-cell PCR, reselection of colonies following second round of G418 selection increased the rate of transgene integration per each colony. These results suggest that transfection with electroporation is the most efficient and the second rounds of G418 selection may be an effective method for transfection of porcine fetal fibroblast cells.
Hong-Rye Kim,Rong-Xun Han,Hye-Ran Lee,Jong-Taek Yoon,Hee-Tae Cheong,Chang-Sik Park,Dong-Il Jin 한국동물생명공학회(구 한국동물번식학회) 2007 Reproductive & developmental biology Vol.31 No.2
The objective of this study was to analyzed pattern of proteins expression abnormally in cloned bovine placenta. TIMP-2 protein whose function is related to extracellular matrix degradation and tissue remodeling processes was one of differentially up-regulated proteins in SCNT placenta. And one of down-regulated protein in SCNT placenta was identified as vimentin protein that is presumed to stabilize the architecture of the cytoplasm. The expression patterns of these proteins were validated by Western blotting. To evaluate how regulatory loci of TIMP-2 and vimentin genes was programmed reprogramming in cloned placenta, the status of DNA methylation in the promoter region of TIMP-2 and vimentin genes was analyzed by sodium Bisulfite mapping. The DNA methylation results showed that there was not difference in methylation pattern of TIMP-2 and vimentin loci between cloned and normal placenta. Histone H3 acetylation state of the nucleosome was analyzed in the cloned placental and normal placenta by Western blotting. A small portion of the protein lysates were subjected to Western blotting with the antibodies against anti acetyl-Histone H3. Overall histone H3 acetylation state of SCNT placenta was significantly higher than those of normal placenta cells. It is postulated that cloned placenta at the end of gestation seems to be unusual in function and morphology of placenta via improper expression of TIMP-2 and vimentin by abnormal acetylation states of cloned genome.
A two-dimensional electrophoresis reference map for the bovine placenta during late pregnancy
Kim, Hong Rye,Han, Rong Xun,Yoon, Jong Taek,Park, Chang Sik,Jin, Dong Il WILEY-VCH Verlag 2010 Proteomics Vol.10 No.3
<P>An understanding of bovine placental gene expression is essential for the study of animal reproductive physiology. Recent reports have found that placental abnormalities occur frequently in cloned bovines and mice. However, the molecular mechanisms underlying bovine placenta function remain unclear. Here, we present a preliminary description of the bovine placenta proteome. Proteins within the isoelectric point ranging from 4.0 to 7.0 and 6.0 to 9.0 were analyzed separately using 2-DE, using three replicates of bovine placenta. Approximately 2000 spots were detected in a placental 2-D gel stained with Coomassie blue. Subsequent excision of 380 spots from gels and MALDI-TOF MS analysis allowed the identification of 273 proteins. Our results revealed the composite profiles of key proteins in the bovine placenta during late pregnancy. These protein profiles will shed light on placental function during pregnancy and assist with functional analysis of the proteins.</P>
Kim, Hong-Rye,Han, Rong-Xun,Lee, Hye-Ran,Yoon, Jong-Taek,Cheong, Hee-Tae,Park, Chang-Sik,Jin, Dong-Il 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10
The objective of this study was to analyzed pattern of proteins expression abnormally in cloned bovine placenta. TIMP-2 protein whose function is related to extracellular matrix degradation and tissue remodeling processes was one of differentially up-regulated proteins in SCNT placenta. And one of down-regulated protein in SCNT placenta was identified as vimentin protein that is presumed to stabilize the architecture of the cytoplasm. The expression patterns of these proteins were validated by Western blotting. To evaluate how regulatory loci of TIMP-2 and vimentin genes was programmed reprogramming in cloned placenta, the status of DNA methylation in the promoter region of TIMP-2 and vimentin genes was analyzed by sodium Bisulfite mapping. The DNA methylation results showed that there was not difference in methylation pattern of TIMP-2 and vimentin loci between cloned and normal placenta. Histone H3 acetylation state of the nucleosome was analyzed in the cloned placental and normal placenta by Western blotting. A small portion of the protein lysates were subjected to Western blotting with the antibodies against anti acetyl-Histone H3. Overall histone H3 acetylation state of SCNT placenta was significantly higher than those of normal placenta cells. It is postulated that cloned placenta at the end of gestation seems to be unusual in function and morphology of placenta via improper expression of TIMP-2 and vimentin by abnormal acetylation states of cloned genome.
Epigenetic characterization of the PBEF and TIMP-2 genes in the developing placentae of normal mice
( Hong Rye Kim ),( Rong Xun Han ),( Yun Fei Diao ),( Chang Sik Park ),( Dong Il Jin ) 생화학분자생물학회(구 한국생화학분자생물학회) 2011 BMB Reports Vol.44 No.8
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. [BMB reports 2011; 44(8): 535-540]
Protein Expression of Mouse Uterus in Post-Implantation
Kim, Hong-Rye,Han, Rong-Xun,Kim, Myung-Youn,Diao, Yunfei,Park, Chang-Sik,Jin, Dong-Il The Korean Society of Animal Reproduction 2009 Reproductive & developmental biology Vol.33 No.4
Pregnancy is a unique event in which a fetus develops in the uterus despite being genetically and immunologically different from the mother, and the underlying mechanisms remain poorly understood. To analyze the differential gene expression profiles in nonpregnant and 7 days post coitus (dpc) pregnant uterus of mice, we performed a global proteomic study by 2-D gel electrophoresis (2-DE) and MALDI-TOF-MS. The uterine proteins were separated using 2-DE, Approximately 1,000 spots were detected on staining with Coomassie brilliant blue. An image analysis using Melanie III (Swiss Institute for Bioinformatics) was performed to detect variations in protein spots between pregnant and nonpregnant uterus. Twenty-one spots were identified as differentially expressed proteins, of which 10 were up-regulated proteins such as alpha-fetoprotein, chloride intracellular channel 1, transgelin, heat-shock protein beta-1, and carbonic anhydrase II, while 11 were down-regulated proteins such as X-box binding protein, glutathione S-transferase omega 1, olfactory receptor Olfr204, and metalloproteinase-disintegrin domain containing protein TECADAM. Most of the identified proteins appeared to be related with catabolism, cell growth, metabolism, regulation, cell protection, protein repair, or protection. Our results uncovered key proteins of mouse uterus involved in pregnancy.