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초자화 냉동법으로 냉동.해동한 Neonatal 생쥐 난소의 생체내 동소이식 후 난포 발달에 관한 연구
이경아,이숙현,윤세진,고정재,차광열,Lee, K.A.,Lee, S.H.,Yoon, S.J.,Ko, J.J.,Cha, K.Y. 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.2
Ovarian development of the vitrified neonatal ovaries after orthotopical transplantation into the ovariectomized adult recipient mouse were observed. Ovaries were collected from the neonatal females on day of birth and grouped for fresh, vitrification for 1-minute, and 3-minute. Vitrified and thawed neonatal ovaries were orthotopically transplanted into ovarian bursa of the adult mice from which endogenous ovaries have removed just prior to the transplantation (1 minute: n=25; 3 minutes n=23). Fresh ovarian tissue transplanted (n=25) mice were included as control groups. Returning of the estrus cycles and the survival and development of the transplanted ovaries were evaluated. Intact ovaries from neonatal, and four weeks old mice were used for comparison of the ovarian development as in vivo-developed control. From 2 weeks after transplantation, 64%, 36%, and 75% of the transplanted mice showed return of the estrus cycles in fresh, 1-minute, and 3-minute groups, respectively. Four weeks after transplantation, all mice were sacrificed and ovarian tissues were recovered for histological analysis. 57.1%, 33.3%, and 64.7% mice in fresh, 1-minute, and 3-minute groups, respectively, had survived ovaries with follicles at various stages of growth from primordial to preovulatory follicles. Corpus lutea were also observed. Results of the present study suggest that 1) normal folliculogenesis has initiated in vivo after vitrification, and 2) the vitrification may be used as a preservation method for ovarian tissues for establishment of ovarian tissue bank.
사람 성장호르몬이 유선조직에서 특이적으로 발현되는 형질전환 생쥐의 개발
유대열(D . Y . Yu),이철상(C . S . Lee),강만종(M . J . Kang),한용만(Y . M . Han),이경광(K . K . Lee) 한국축산학회 1993 한국축산학회지 Vol.35 No.1
In an attempt to develop transgenic animals producing biologically active proteins into the milk., expression vectors (pC and pCS) were constructed. The pC vector consists of the promoter 2.8-kb DNA fragment of rat ?-casein gene and pSP70 plasmid. The pCS vector contains the SV40 poly(A) sequences in the multiple cloning sites of pC vector. Human growth hormone gene was then inserted into the cloning site of each vector, which was designated to pChGH and pCShGH. The plasmids were injected into one-cell pronuclear mouse embryos as a purified Xhol/Hpal fragments containing no procaryotic sequences. The injected embryos were implanted into pseudopregnant females, and 117 mice were born. The live of them were identified as being transgenic (ChGH line: 2 mice, CShGH line: 3 mice) by diagnostic Southern blot hybrization with human growth hormone gene. Among them, three transgenic mice transmitted the DNAs integrated on their chromosomes to their progenies and secreted human growth hormone in milk at the level of 160 ng/㎖ (ChGH-2-2 line), 80 ng/㎖ (CShGH-1 line), and 2-4 ng/㎖ (CShGH-3 line), respectively. The above cases show that no human growth hormone was detected in the serum, and these results suggest that the inserted gene is specifically expressed in the mammary gland of the transgenic mice by the regulation of the β-casein promoter.
이경아,이숙현,하상덕,윤세진,고정재,이우식,윤태기,차광열,Lee, K.A.,Lee, S.H.,Ha, S.D.,Yoon, S.J.,Ko, J.J.,Lee, W.S.,Yoon, T.K.,Cha, K.Y. 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.2
The present study was conducted to evaluate whether vitrification could be used for ovarian tissue preservation. The important issue here is that the vitrification is very simple, easy, and economical compared to the conventional cryopreserving method that using automatic freezing instrument. Human ovarian cortical tissues were cryopreserved by vitrification with 5.5 M ethylene glycol and 1.0 M sucrose as cryoprotectant. Three points of temperature ($4^{\circ}C$, room temperature, and $37^{\circ}C$) and two points of duration (5 or 10 minutes) for cryoprotectant treatment were examined to determine the best condition for vitrification of the human ovarian cortical tissues. After thawing, viability of the isolated primordial follicles was examined by dye-exclusion method. Histological appearance of tissues before and after the cryopreservation was evaluated. There was no toxic effect of the 5.5 M ethylene glycol on the primordial follicles. However, when the tissues were treated with cryoprotectant at $37^{\circ}C$ for 10 minutes and exposed to liquid nitrogen, it seems likely that there is certain deleterious effects on the viability of the primordial follicles. The highest viability of the primordial follicles was obtained with the treatment of cryoprotectant at room temperature for 10 minutes. Follicles and oocytes survived after freezing and thawing had the similar normal shapes as was seen in the specimens before cryopreservation. It would be useful to apply vitrification in establishing ovarian tissue banking for clinical purposes.
Transgenic 생쥐생산에 있어서 미세주입시기 및 외래유전자의 농도가 삽입빈도에 미치는 영향
한용만(Y . M . Han),이철상(C . S . Lee),강만종(M . J . Kang),유대열(D . Y . Yu),이경광(K . K . Lee) 한국축산학회 1990 한국축산학회지 Vol.32 No.6
The present study was undertaken in an attepmt to improve the efficiency of integration frequency in transgenic animals. In order to examine the effect of microinjection time on integration frequency, foreign DNA was microinjected into the male pronucleus of each fertilized egg at 2hr intervals from 21 to 27hr after HCG injection. The results revealed that the overall integration efficiency was somewhat higher in the group of 23-25hr than in the other groups. Wealso investigated on the effect of DNA concentrations(2, 4, 8 & 20ng/ul) on integration frequency, but differences between experimental groups were not statistically significant.
사람 다수정난자의 체외배양시 Fragmented Embryo와 Non-fragmented Embryo에서의 Methionine 유입량 및 미토콘드리아 분포양상의 비교
도병록,정미경,장미경,이경아,고정재,윤태기,차광열,Do, B.R.,Chung, M.K.,Chang, M.K.,Lee, K.A.,Ko, J.J.,Yoon, T.K.,Cha, K.Y. 대한생식의학회 1995 Clinical and Experimental Reproductive Medicine Vol.22 No.3
Despite the frequent incidence of embryo fragmentation in early human embryos, the reason of the embryo fragmentation has not been known yet. This study was conducted to investigate the histological difference(s) between fragmented (FR) and non-fragmented (NFR) human embryos focusing on comparison of mitochondrial distribution and protein synthesis. Multi-pronuclei zygotes (MPZ) such as three or more pronuclei containing in human in vitro fertilization and embryo transfer (IVF-ET) program were used for this study. MPZ were cultured in TCM-199 supplemented with 10% of human fetal cord serum (hFCS) in 5% $CO_2$ incubator at $37^{\circ}C$ for 24 hours. The cleaved embryos to 2-4 cells after 24 hours were grouped by their grade of fragmentation. Embryos were stained with Rhodamine123 (Rh123) and fluorescence was evaluated under the fluorescence microscope through PB 450-490 filter (Leitz). Regarding to protein synthesis during early human embryogenesis, there is no significant difference in the amount of synthetic proteins between FR and NFR embryos. Distribution of cytoplasmic organelles in embryos was evaluated by transmission electron microscope (TEM). The cytoplasmic distribution of mitochondria was different between FR and NFR embryos. The mitochondrial distribution was even in NFR, whereas severely aggregated in FR. It is not able to clarify in the present study whether this uneven mitochondrial distribution in FR embryo is the reason for embryo fragmentation or is the result from fragmentation. Physiological disparity related to the mitochondrial distribution may be one of the reasons for embryo fragmentation. Further studies should be addressed to investigate the physiological differences between FR and NFR embryos.
성숙배양액에 첨가하는 인간체액 (Human Body Fluids) 및 성선자극호르몬이 생쥐 미성숙난자의 핵성숙과 수정능력에 미치는 영향
박기상,손원영,김진희,이경아,한세열,고정재,차광열,Park, K.S.,Son, W.Y.,Kim, J.H.,Lee, K.A.,Han, S.Y.,Ko, J.J.,Cha, K.Y. 대한생식의학회 1994 Clinical and Experimental Reproductive Medicine Vol.21 No.2
Purpose of the present study was to find the optimal culture conditions for the maturation and fertilization of immature oocytes by the use human body fluids and gonadotropins (Gn) in the mouse model. Cumulus-enclosed mouse immature oocytes were incubated in the medium containing various human body fluids with or without Gn in vitro, and examined to confirm nuclear maturation (NM) and fertilization. Female ICR mice were stimulated with 7.5 IU pregnant mares' serum gonadotropin (PMSG). Cumulus-enclosed immature oocytes were isolated at 48-52 hr post PMSG injection and cultured in TCM 199 supplemented with various concentrations (20, 50, and 70%) of human body fluids such as fetal cord serum (hCS), follicular fluid (hFF), peritoneal fluid (hPF) and amniotic fluid (hAF) in the presence or absence of 10 IU/ml PMSG and 10 IU/ml human chorionic gonadotropin (hCG) for 18 hr. Fetal calf serum (FCS) was used as a control for the supplements. Matured oocytes were fertilized with sperm collected from the epididymis of male mice. Fertilization was conducted in T6 medium containing 15 mgl ml bovine serum albumin, and confirmed at 6 hr post-insemination. Evaluation of nucler maturation and fertilization was carried out by rapid staining using fuchin. There was no significant difference between the effects of human body fluids and FCS supplements on nuclear maturation of cumulus enclosed mouse immature oocytes. When maturation medium was supplemented with 20% hPF or 20% hAF, fertilization rates were significantly (P<0.01) lower than that of 20% FCS, hCS and hFF groups. However, higher concentrations of body fluids during IVM were not more beneficial on fertilizability of oocytes. The addition of Gn significantly increased the fertilization rates in hPF and hAF groups (hPF without Gn; 51.5%, compared with 85.1% for addition of Gn, and hAF without Gn; 30.1% compared with 85.8% for addition of Gn) at 20% concentration. These results suggest that human body fluids at 20% concentration and gonadotropins can be used as supplements for the maturation of mouse immature oocytes in vitro. When gonadotropins supplemented with the human body fluids in the maturation medium, fertilizability of mouse immature oocytes was increased in hPF and hAF groups. These results can be applied to maturation of human immature oocytes in vitro.
유대열,이경광 中央大學校 遺傳工學硏究所 1993 遺傳工學硏究論集 Vol.6 No.1
In 1980's transgenic animals expressing growth hormone genes derived from various species were developed to improved growth of livestocks or feed efficiency. But they resulted in many side effects because the constitutive and over expression of the genes affected the endocrine system. To solve these problems, other approaches using peptides related with muscle differentiation were tried but it has so far been unsuccessful. Recently, transgenic technology has been applied to develop livestocks which secrete large amounts of foreign proteins into mammary glands. Animal bioreactor is one of the most promising approaches applicable to industry. Although some transgenic animals show side effects like lactational shutdown, the animal bioreactor system is hopeful if we develop efficient expression vectors and get the basic information for the item to be produced. Basic knowledge and various technologies, including gene cloning, recombinant DNA, in vitro cell culture micromanupulation of embryos, cryopreservation, embryo transfer, breeding of animals, should be put together on line to improve the productivity of transgenic animals.