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돼지 단위생식란의 세포사멸 유전자 발현 양상에 관한 연구
손종윤,김상환,정덕원,류춘열,윤종택 한국수정란이식학회 2015 한국동물생명공학회지 Vol.30 No.3
The nature of molecular mechanisms governing embryonic cell block is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to determine effects of programmed cell death on porcine oocytes development in vitro after parthenogenesis. Among the blastocysts matured in 3MA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of Cyst < 3MA < RP. However, Casp-3 and TNF-r RNA gene expression level decreased in the order of RP < 3MA < Cyst. Expression of mTOR within the RP-cultured blastocyst was the most highly to the inner cell mass, while 3MA-cultured blastocyst showed very lowest expression in inner cell mass. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. When the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each RP treatment group, with the level of another treatment group being relatively higher. Analyses of TIMP-2 and TIMP-3 revealed that their expression was higher in groups that did not receive RP treatment. More specifically, the level of TIMP-2 was not affected by Cyst treatment, while the level of TIMP-3 was higher in 3MA and RP treatment group. There was highly cell division activation efficiency of parthenogenesis on cultured system of RP supplement IVC medium. Therefore, these results suggest that embryo development was significantly increased in conditional culture medium with active autophagy as compared to common cultured condition. Further investigation of this distinction may enable the development of innovative improvements for the production of porcine somatic cell nuclear transfer.
칡소의 MC1R의 유전자형에 따른 교배 조합이 자손의 모색과 유전자형 변이에 미치는 영향
김상환,정경섭,이호준,백준석,정덕원,김대은,윤종택 韓國受精卵移植學會 2013 한국동물생명공학회지 Vol.28 No.3
Bovine coat color is decided by the melanocortin receptor 1 (MC1R) genotype mutation and melanogenesis. Specially, in the various cattle breeds, dominant black coat color is expressed by dominant genotype of ED, red or brown is expressed in the frame shift mutation of recessive homozygous e by base pair deletion and wild type of E+ is expressed in various coat colors. However, not very well known about the effected of MC1R genotype mutation on the coat color through family lines in KBC. Therefore, this study were to investigate effect of MC1R genotype mutation on the coat color, and to suggest mating breed system in accordance with of MC1R genotype for increased on brindle coat color appearance. Parents (sire 2 heads and dam 3 heads) and offspring (total : 54 heads) from crossbreeding in KBC family line with the MC1R genotype and phenotype records were selected as experimental animals. The relationship between melanocortin 1 receptor (MC1R) genotypes expression verified by PCR-RFLP, and brindle coat color appearance to the family line of the cross mating breed from MC1R genotype pattern was determined. As a result, 4MC1R genetic variations, E+/E+ (sire 1), E+/e (sire 2 and dam 3), E+/e with 4 bands of 174, 207 and 328 bp (dam 1) and E+/e with 3 bands of 174, 207, 328 and 535 bp (dam 2) from parents (sire and dam) of KBC. However, 3 genetic variations, e/e (24%), E+/E+ (22%) and E+/e (56%) were identified in offspring. Also, brindle coat color expressrated was the e/e with the 0%, E+/E+ with 67% and E+/e with 77% from MC1R genotype in offspring on the cross mating of KBC. Furthermore, when the sire had E+/e genotype and the dam had E+/E+ with the 3 bands or E+/e genotype, and both had whole body-brindle coat color, 62% of the offspring had whole body-brindle coat color. Therefore, the seresults, the mating system from MC1R genotype patterns of the sires (E+/e) and dams (E+/E+ with the 3 bands or E+/e) with brindle coat color may have the highest whole body-brindle coat color expression in their offspring.
김상환,정덕원,이호준,황수연,민관식,윤종택 한국동물생명공학회(구 한국동물번식학회) 2009 Reproductive & developmental biology Vol.33 No.1
This study was conducted to investigate the specific expression genes in the cloned bovine tissues. Donor cells, cloned tissues were analysed by RAPD-RFLP method. The results were detected three genes (CH-U7B, CH-U7M and CH-U7P) in the cloned fetus. It was found a single copy genes by southern hybridization. Sequence analysis of CH-U7M gene was shown 99% homology to a previously reported EST from a cloned bovine fetus. The putative ORF was encode a protein of hydrophobicity index 0.03. Semi-quantitative RT-PCR by using the CH-LS001 specific primer was remarkably detected in the lung tissue of cloned fetus. Further investigation of these genes may provide one of the key information to explain the early death, abnormal fetus, large off-spring and the low pregnancy rate in the production of cloned bovine.
NCSU 23 배양액에 첨가된 Insulin-Like Growth Factor-1(IGF-1)이 돼지 수정란의 체외발육에 미치는 영향
김수,남동현,정연우,김혜수,이소현,이갑상,현상환,김대영,강성근 韓國受精卵移植學會 2002 한국동물생명공학회지 Vol.17 No.3
본 연구는 돼지 체외수정란의 배양시스템의 최적화를 위하여 수행되었다. 기본배양액으로는 NC-SU-23을 사용하였으며, 실험 1에서는 IGF-I이 각각 0, 1, 10, 100 ng/의 농도로 첨가된 배양액에서 배양하여 분할율, 배반포 발달율과총 세포수를 확인하였다. 농도 경사에 따른 분할율은 차이가 없었으나(65.5%~69.5%), 배반포 발달율에 있어서는 10 ng/의 농도로 첨가하였을 경우가 가장 높았다(P<0.05). 실험 2는 배 아 유전자 활성 This experiments were conducted to examine the effect of Insulin-Like Growth Factor-1 (IGF-1) on preimplantation development of porcine IVF embryos. Cumulus-oocyte complexes (COCs) were matured and fertilized in vitro, and their development was monitored for 168h pi(post-insemination). In Experiment 1, embryos were cultured in NCSU23 supplemented with various concentration of IGF-1(0, 1, 10, 100 ng/). At 168h, blastocysts' cell number of inner cell mass and trophectoderm were counted. More(P<0.05) blastocysts were cultured in the medium supplemented with 10ng/m1 of IGF-1 and total cell number was the highest when embryos were cultured(P<0.05) in the same IGF-1 concentration. In Experiment 2, one-cell embryos were cultured in medium supplemented as follows: I) no supplementation; 2) without IGF-1 for 48 h pi(post-insemination) and with IGF-1 for subsequent 120 h: 3) IGF-t for 168 h; 4) with IGF-1 for 48 h and without IGF-1 for 120 h. Compared with other treatment groups, a significant increase in the proportion 2-cell embryos(P<0.05) and blastocyst(P<0.05) were obtained when cultured in medium supplemented with IGF-1 throughout in vitro culture for 168 h. As results, blastocysts cell number was the highest when oocytes were cultured in medium supplemented with 10 ng/ IGF-I throughout culture Period. However no significant difference was found on developmental competence when IGF-I was added at 4-cell stage embryos. In conclusion, 10 ng/ml of ICF-1 supplemented in NCSU-23 medium enhances porcine IVP embryo development to the blastocyst stage, but does not significantly increase cell number of blastocysts.
소 수정란의 할구 분리방법에 따른 생존율 및 성판별 PCR의 개선
김상환,김경래,이호준,정덕원,김대은,이득환,윤종택 韓國受精卵移植學會 2013 한국동물생명공학회지 Vol.28 No.3
The present study was conducted to compare on embryo survival rates by blastomere isolation methods, and establish the optimal PCR procedure for perform the sexing of bovine blastocysts produced by IVF. IVF embryos used in the study was used the Bisected or Sliced methods for blastomere isolation, and the survival rates of blastocyst with rapid way of sexing PCR was assessed. In the present study for survival rates in blastocyst was the total cleavage rate was 75% and a blastocyst development among cleaved embryos was 40%. Survival rate of embryos treated with intact, bisected or sliced method was 100, 63.3 or 81.3%, respectively. Therefore, survival rate of embryos treated with sliced method was higher compared to that of embryos treated with bisected method. The sexing rate of female or male was not significantly different between S4BFBR primer and BSY + BSP primer (1.75 : 1 vs. 1.43 : 1), respectively. Because of the PCR amplification using the S4BFBR primer was simpler method than multiplex PCR amplification method. Furthermore, the accuracy of sexing rate and reduction of PCR work time between 2-step and 3-step of PCR methods was 98.0% / 1.5 hr and 97.0% / 3.5 hr, respectively. Based on these results, it can be suggested that the sliced and PCR methods we developed was very effective method to reduce time consuming and procedure of PCR amplification for sexing with the increase of survival rate on the blastocyst.