수핵 난자의 활성화 방법과 공핵 수정란의 배양체계 및 할구의 크기가 소 핵이식 수정란의 발달에 미치는 영향

심보웅,조성근,이효종,박충생,최상용 한국동물생명공학회(구 한국동물번식학회) 1998 Reproductive & developmental biology Vol.22 No.4

On the Development of Parthenogenetic Oocytes by Cytochalasin B and Production of Cloned Mice by SCNT

심보웅,민관식 사단법인 한국동물생명공학회 2014 한국동물생명공학회지 Vol.29 No.2

This study was conducted to optimize the efficiency of cloning and to produce cloned mice. The majority of clonedmammals derived by nuclear transfer (NT) die during gestation and have enlarged and dysfunctional placentas. In thisstudy, the optimized conditions were established to produce clone mice. The parthenogenetic oocytes were activatedafter 6 h regardless of cytochalasin B (CB) concentration. CB treatment (2 μg/ml) was found second polar body. Lower concentration of CB was decreased the activation rate, but the second polar body was the best highly increasedduring 6 h incubation. The small fragments were exhibited in the 5 μg/ml treatment of CB, but it was not found inlower concentration groups (> 2.5 μg/ml). To examine effects of SrCl2 on the adult cumulus cells, somatic cell NToocytes were exposed during 0.5, 1 and 6 hrs. The second polar body was significantly greater in 0.5 h exposuregroup (6.6%) than 1, 6 hrs. Developmental rate from 2-cell to 4-cell was the lowest in 7.5 mM Strontium chloride(SrCl2) groups (84.1% and 64.3%) than 5, 10 m MSrCl2. The implantation rate was not significantly difference among5, 7.5 and 10 m MSrCl2 group. Three live fetuses were produced by SCNT. SCNT placentas were remarkably heavierthan IVF group (8 fetuses) (0.34, 0.34, 0.33 vs 0.14 g) compared with the placenta weight of IVF and SCNT clones.

배양액이 마우스 핵이식 재구축배의 초기발생에 미치는 영향

심보웅,권오용,이은화,김순희,서정선 한국수정란이식학회 2002 한국수정란이식학회 학술대회 Vol.2002 No.1

본 실험은 Piezo-미세조작기(PrimeTech Ltd., Japan)를 사용하여 마우스 핵이식 후 재구축배를 CZB와 KSOM 두가지 배양액을 사용하여 체외배양성적을 비교 검토하였다. MII의 미수정란은 성숙한 4~5주령 B6D2Fl에 hCG 주사 후 14시간째에 과적 방법을 통해 난관의 팽대부로 부터 회수하였고, metaphase II chromosome-spindle complex와 최소량의 세포질을 내경이 10인 피펫으로 흡입하여 탈핵하였다. 핵이식에 사용된 난구세포(8-l0)는 3시간동안 12% PVP 에처리 하여 piezo-미세조작기를 이용하여 세포질에 세포의 핵을 직접 미세주입 하였다. 핵이식 후 생존한 재구축배는 2시간동안 배양한 후 10mM SrC1와 5/의 cytochalasin B가 첨가된 -free CZB에서 6시간 활성화 처리하였다, 활성화 처리 후 위전핵이 관찰된 재구축란을 CZB 와 KSOM 배지에서 배양하면서 발달률을 비교하였고, 상실배 및 배반포배로 발달한 재구축배를 day 3 대리모에 이식하였다. 표 1에서 보는 바와 같이 재구축배의 2-cell로의 발달률에 있어서 KSOM이 CZB에 비하여 유의적으로 높게 나타났으며(P<0.05), 또한 4-cell과 상실배/배반 포배로의 발달률에 있어서도 KSOM이 CZB에 비하여 유의적으로 높은 발달률을 나타내었다(P<0.01). 또한 KSOM 배지에서 배양된 상실배/배반포배를 대리모에 이식한 경우에 11.5 d.p.c에 생존한 태아가 관찰되었다. 이상의 결과로 핵이식 재구축배의 활성화 처리 후의 발생에는 KSOM 배지가 CZB 배지에 비하여 유효함을 확인 할 수 있었다.그와 같은 배양 기술을 이용하여 외래유전자를 도입한 일련의 결과에 관하여 보고 하고자한다., 이것은 세포내 유전자가 transfection되지 않은 세포도 neo selection에서 선발된다는 것을 제시하고 있다. 따라서 체세포를 이용한 형질전환동물 생산을 위해서는 세포내 유전자 도입과 선발 과정에서 나타난 colony에 대하여 보다 엄격한 screen을 하는 것이 필요한 것으로 생각된다.로 우점하였다. 여름철 식물플랑크톤 대발생에 영향은 수온과 직산염이 중요하였으나, 부유물질 크게 기여하지 못하였다.애를 확인하고 지도 관점을 파악하는 것을 포함한다. 그러나 본 논문은 역사발생적 수학 학습-지도 원리의 실제적인 적용에 관하여는 기초적인 연구에 지나지 않기 때문에, 역사발생적 원리를 학교수학에 실제적으로 적용하기 위해서는 각각의 내용에 대한 철저한 역사적 분석을 바탕으로 하는 후속 연구가 필요하다./TEX>구성교육이 조선총독부의 관리하에서 실행되었다는 것을, 당시의 사범학교를 중심으로 한 교육조직을 기술한 문헌에 의해 규명시켰다. nd of letter design which represents -natural objects and was popular at the time of Yukjo Dynasty, and there are some documents of that period left both in Japan and Korea. "Hyojedo" in Korea is supposed to have been influenced by the letter design. Asite- is also considered to have been "Japanese Letter Jobcheso." Therefore, the purpose of this study is to look into the origin of the letter designs in t

Production of cloned mice by aggregation of tetraploid embryo

심보웅,민관식 한국통합생물학회 2014 Animal cells and systems Vol.18 No.5

Mouse chimeras can also successfully be produced using tetraploid host embryos. This study was conducted to optimize theefficiency of cloning and to produce cloned mice using tetraploid host embryos. Six hours of activation with strontium(SrCl2) was optimal for somatic cell nuclear transfer (SCNT) embryos. Cytochalasin B (CB) concentration (5 μg/ml) duringenucleation was evaluated in the efficiency of implantation sites and fetus offspring. Continuous exposure to 5–50 nMtrichostatin A (TSA), a histone-deacetylase inhibitor (HDACi), for 10 h is recommended for production of clone mice. Aggregated SCNTs were constructed by aggregation of SCNT embryos with tetraploid embryos to reduce epigenetic errorsin the placenta. The pregnancy and implantation rates of aggregated SCNT were significantly higher than those of SCNTalone. The full-term developmental rate of aggregated embryos was also higher than that of SCNT (3.57 vs. 1.16). Theplacental weight of SCNT clones was significantly higher than that from in vitro fertilization (IVF). However, the placentaweight of aggregated SCNT clones was nearly the same as that of embryos in the IVF group. The placentas of SCNT-onlyclones appeared to have the hyperplastic histology typical of mouse clones. We produced a total of 36 clone mice, includingnine heads derived from aggregated SCNT. One-half of clones derived from aggregated SCNT survived to adulthood, and14-clones derived from SCNT grew into healthy adults. The aggregated SCNT method was useful for significantly reducingthe placental weight of cloned mice and improving the efficiency of SCNT.

Cadmium exposure impairs porcine embryonic development by inducing oxidative stress and mitochondrial dysfunction

김민주,전세빈,강효구,송봉석,심보웅,김선욱,정필수,조성근 사단법인 한국동물생명공학회 2024 Journal of Animal Reproduction and Biotechnology Vol.39 No.1

Background: Cadmium (Cd) is toxic heavy metal that accumulates in organisms after passing through their respiratory and digestive tracts. Although several studies have reported the toxic effects of Cd exposure on human health, its role in embryonic development during preimplantation stage remains unclear. We investigated the effects of Cd on porcine embryonic development and elucidated the mechanism. Methods: We cultured parthenogenetic embryos in media treated with 0, 20, 40, or 60 μM Cd for 6 days and evaluated the rates of cleavage and blastocyst formation. To investigate the mechanism of Cd toxicity, we examined intracellular reactive oxygen species (ROS) and glutathione (GSH) levels. Moreover, we examined mitochondrial content, membrane potential, and ROS. Results: Cleavage and blastocyst formation rates began to decrease significantly in the 40 μM Cd group compared with the control. During post-blastulation, development was significantly delayed in the Cd group. Cd exposure significantly decreased cell number and increased apoptosis rate compared with the control. Embryos exposed to Cd had significantly higher ROS and lower GSH levels, as well as lower expression of antioxidant enzymes, compared with the control. Moreover, embryos exposed to Cd exhibited a significant decrease in mitochondrial content, mitochondrial membrane potential, and expression of mitochondrial genes and an increase in mitochondrial ROS compared to the control. Conclusions: We demonstrated that Cd exposure impairs porcine embryonic development by inducing oxidative stress and mitochondrial dysfunction. Our findings provide insights into the toxicity of Cd exposure on mammalian embryonic development and highlight the importance of preventing Cd pollution.

한우에서 초음파 유래와 도축장 유래 난포란을 이용한 체외 수정란 생산에 관한 연구

손우진,강태영,조성근,심보웅,최민철,최상용,박충생,이효종 韓國受精卵移植學會 1998 한국동물생명공학회지 Vol.13 No.2

The present study was carried out for the comparative study on the collection of bovine follicular oocytes by ultrasound-guided ovum pick-up(OPU) and slaughterhouse-derived (SHD) ovary aspiration and in vitro production of bovine embryos with the follicular oncytes in Korean native cows. Bovine follicular nocytes were observed with a 6.5 MHz convex-array ultrasound transducer designed for intravaginal use and the oocytes were collected with the aspiration equipment attached to the ultrasonograph. Bovine ovaries were collected and transported in phosphate buffered saline from the local slaughterhouse, the follicular oocytes were collected by the aspiration method. The collected follicular oocytes in good quality were matured, fertilized and cultured in the media. The total number of the visible follicles and the recovery rate of follicular oocytes were increased in ultrasonography following follicle stimulating hormone(FSH) treatment in Korean native cows. The mean recovery rate of oocytes was 66.2, 52.8 and 41.7% in the FSH-OPU, non-treatment-OPU and SHD ovaries, respectively. The mean number of recorved oocytes per cow were not significantly(P<0.05) different between the FSH-OPU(14.011.54) and SHD(17.1i6.21) groups, but the numbers in both groups were significantly(P<0.05) higher than the number in the non-treatment-OPU(3.71.57) group. The mean number of usable nocytes in Grade T /11 per ovary was 6.3, 4.8 and 1.3 in the cows of the SHD, FSH-OPU and non-treatment-OPU groups, respectively. The in vitro developmental rate to the blastocyst was not significantly different between the oocytes obtained via OPU(37.1%) and SHD(29.3%). Therefore, the ultrasound-guided OPU technique can be applied to the production of excellent embryos from the high-quality cows, and for the large scale production of in vitro bovine oocytes and embryos, the SHD ovary aspiration method is valuable.

토끼의 정상 및 핵이식배의 유리화 및 완만동결에 따른 융해 후 발달율

강다원,최창용,하란조,강태영,심보웅,최상용,이효종,박충생 韓國受精卵移植學會 1998 한국동물생명공학회지 Vol.13 No.1

In order to improve the cryopreservation by vitrification or slow freezing of nuclear transplant rabbit embryos, the effects of factors affecting embryo cryopreservation such as cryoprotectants, equilibration, cooling rate and post-thaw dilution on post-thaw survial and development were determined using intact embryos of morular stage. And the post-thaw development of nuclear transplanted embryos cryopreserved under the optimal conditions examined was compared between vitrification and slow freezing. The cryoprotectant solution used was ethyleneglycol-ficoll-sucrose (EFS) or ethyleneglycol-poly-vinylpyrrolidone-galactose- I (EPG- I ) for vitrification, and EPG- II for slow freezing. To examine the viability of frozen-thawed embryos, the nuclear transplanted embryos were co-cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) for 24 hrs and the intact morulae were co-cultured with BOEC for 5 days and 3 days to hatching blastocyst stage in 39 ºC 5% incubator. The results obtained were as follows: Following vitrification with EFS, the post-thaw development of rabbit morulae to hatching blastocyst was significantly(P<0.05) higher in compacted stage(82.4%) than in early morular stage(60.0%). The post-thaw development of compacted morulae to hatching blastocyst was similarly high in vitrification with EFS(82.4%), EPG- I (85.0%) and in slow freezing with EPG- II (83.3%). Following vitrification with EPG- I, the post-thaw development of intact rabbit morulae to hatching blastocyst was similar as 78.0% and 85.0% in 1-step and 2-step post-thaw dilution, respectively. The post-thaw development of nuclear transplanted rabbit embryos of compacted morulae stage to hatching blastocyst was similarly 43.6% and 40.0% in vitrification with EPG- Iand slow freezing with EPG- II, respectively. These results indicated that the rabbit nuclear transplant and intact embryos of morulae stage could be well cryopreserved with either vitrification or slow freezing procedure.

유리화와 완만동결법에 의한 토끼 정상 및 핵이식배의 동결보존후 배발달율

강다원,강태영,최창용,하란조,심보웅,공일근,박충생,최상용,이효종 한국수정란이식학회 1997 한국수정란이식학회 학술대회 Vol.1997 No.2

젖소의 초음파 유도에 의한 난포란의 채란 및 체외수정란의 생산

박충생,조성근,박준규,심보웅,강태영,손우진,최민철,이효종,최상용 한국수정란이식학회 1997 한국수정란이식학회 학술대회 Vol.1997 No.2

한우 체외수정란을 이용한 쌍자생산에 관한 연구

송상현,조성근,조상래,심보웅,강다원,정기화,손동수,이효종,박충생 韓國受精卵移植學會 2000 한국동물생명공학회지 Vol.15 No.1

This study was carried out to improve a technique of embryo transfer for twin calves production in Hanwoo cattle. Blastocysts for the donor of embryo transfer were classified into three criteria by accessment of morphology; early blastocyst, blastocyst and expanded blastocyst. Tow embryos were introduced transcervically into utrerine horn either of Hanwoo or Holstein by ipsilaterally or contralaterally to the corpus luteum. Thiry-six out of 57 recipients cows were inseminated by artificially on the next day of estrus, and followed by transfer of embryos into contralaterally. The pregnance rates of recipients following transfer of bovine embryos of day 7, 8 and 9 was 43.5, 18.2 and 8.3%, respectively. These results appeared that these was a significant (P<0.05) difference between on day-7 embryos and day-9 embryos, but not between on day-8 and day-9 embryos. Although there was not significant(P<0.05) difference in the pregnancy rates between the blastocysts(11/25, 44%) and expanded blastocysts(2/19, 10.5%) and between the blastocysts and early blastocysts(2/13, 15.4%), the embryos at blastocyst stage are more suitable than others for obtaining higher rate of pregnancy. There was no significant difference on pregnancy of the embryos transferred prior to presence(6/21, 29%) or absence (9/36, 25%) of artificial insemination. On pregnancy of Holstein, 2(15.4%) out of 13 recipients were pregnant in heifer. Similar Pregnancy rates were obtained between 1∼2 parities and 3∼4 parities by 30% (6/20) and 27.3%(3/11), respectively. Taken together, there was not significant difference in pregnancy rate due to small number of recipients used for this experiment. Both of Hanwoo and Holstein introduced the embryos by contralsterally to the corpus luteum were slightly higher pregnancy rate compare to by ipsilaterally (12/41, 29.3% vs, 3/16, 18.8%). The ratio of production of twin and single calves in Holstein was 20% (9/45) and 2.2% (1/45), respectively. However, in Hanwoo cows both of production of twin and single were similar as 8%. This result suggests that Holstein as recipients was superior to Hanwoo cows for production of twin calves. Out of all 15 pregnant, 12(80%) were produced a total of 22 normal calves in which the others composed of abnormal, as judging as 2(13.3%) for abortion and 1(6.6%) for stillbirth during the pregnant period.