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이경광(Kyung Kwang Lee),한용만(Yong Mahn Han),남궁욱(Uk Namgung),이철상(Chul Sang Lee),김용주(Yong Joo Kim),김재만(Jae Man Kim),김지영(Ji Young Kim),한문희(Moon Hi Han) 한국축산학회 1989 한국축산학회지 Vol.31 No.3
The structural gene of human growth hormone was fused with mouse MT-1 promoter and the fusion plasmid was designated as pMThGH. The 2.6Kb BamHI/Bst EⅡ DNA fragment of pMThGH was used as a source of human growth hormone fusion gene in these studies. For microinjection, DNA fragments were diluted with TE buffer in the range of 2-8ng/ul. Fertilized one-cell eggs were flushed from the oviducts on 22-24 hours after HCG injection. To remove cumulus cells, cumulus cell masses were treated with hyaluronidase (300units/ml) and then washed 3 times in fresh medium. The obscure pronuclei of eggs due to hybrid(C57BL/6x CBA) mouse were visible after centrifugation for 3 min. at 15,000rpm. Visualization of nuclear structures was aided by the use of a differential interference contrast microscope and microinjection was carried out under x200 magnification using a Leitz micromanipulator. The microinjected eggs were surgically transferred to the oviducts of synchronized recipients. The litters developed from microinjected eggs were weaned about 4-6 weeks after birth and a piece of tail from each mouse was cut off about 1-1.5cm to analyze foreign DNA. The genomic DNAs isolated from each tail were analyzed by dot hybridization and Southern blot to determine which mouse carried MThGH genes. The results obtained in these experiments were summarized as follows; I. Of 106 microinjected eggs cultured for 96 hours in vitro, 53 eggs(50.0%) were developed to normal blastocyst stage. 2. When microinjection was carried out in the medium with or without Cytochalasin B(5 ug/ml), survival rate of eggs was 72.9% and 50.2%, respectively. 3. 382 microinjected eggs were transferred to the oviducts of 23 recipients and 140 mice consequently developed from these eggs. 4. Of 77 mice analyzed by dot hybridization and Southern blot, 6 mice were positive for MThGH genes and each carried at least one copy of hGH gene. 5. Transgenic mice had very high levels of hGH in their serum, 450 - 6, 100mg/ml, and body weights of them were heavier 1.5-2.0 times than those of controls.
햄스터 H-Y항체와 중합효소연쇄반응을 이용한 소 수정란의 성감별
유일정,김용준,이경광,Yu, Il-jeong,Kim, Yong-jun,Lee, Kyung-kwang 대한수의학회 1999 大韓獸醫學會誌 Vol.39 No.1
To determine sex of bovine embryos using hamster histocompatibility Y(H-Y) antibodies, bovine compact morulae were incubated for 6 hours in TCM199 supplemented with 10% hamster H-Y antiserum and the embryos with developmental arrest were diagnosed as male embryos, while the embryos showing development during the incubation as female embryos. This presumptive embryo sexing was confirmed by polymerase chain reaction(PCR)method. 1. In the result of hamster sperm cytotoxicity test to measure H-Y antibody titer, the rate of dead sperm was considerably lower in H-Y antiserum absorbed with hamster male splenocytes than in H-Y antiserum absorbed with hamster female splenocytes or H-Y antiserum unabsorbed with splenocytes(p<0.01). 2. The rate of oocytes fertilized in vitro and the rate of blastocysts of the fertilized oocytes were 58.5% and 32.4%, respectively. The rate of blastocysts on day 8 was 15.9%, denoting the highest rate during whole culture period posterior to in vitro fertilization (IVF). 3. The bovine 16 cell and compact morulae embryos incubated in the medium supplemented with hamster H-Y antibodies showed 37.1% and 48.9% of developmental arrest which were diagnosed as male, respectively, and rates of redeveloped embryos from the arrested were 24.1% in 16 cell and 44.3% in compact morulae embryos, respectively, denoting higher rate of sex determination and rate of redevelopment in compact morulae than 16 cell embryos. 4. Bovine compact morulae of Korean cattle and Holstein were treated with hamster H-Y antibodies for sex determination and the rates of developmental arrest(diagnosed as male) were 48.4% for Korean cattle and 47.9% for Holstein, respectively. The rates of redeveloped embryos to blastocyst after treatment were 42.6% for Korean cattle and 41.8% for Holstein, respectively, showing no significant differences of sex determination and redevelopment between both breed. 5. The sex determination of bovine embryos(Korean cattle and Holstein) using hamster H-Y antibodies was diagnosed by PCR for confirmation, denoting the rates of 86.1% for Korean cattle and 85.9% for Holstein male embryos, respectively, and the rates of 91.9% for Korean cattle and 90.1% for Holstein female embryos, respectively, with no significant differences of sex determination between both breed. These results indicated that hamster H-Y antibodies can be usable for sex determination of bovine embryos of Korean cattle and Holstein, the viability of bovine embryos was sustained while being cultured in the medium supplemented with hamster H-Y antibodies of appropriate titer and sex determination of bovine embryos by PCR can be feasible for confirmation.
사람 성장홀몬 유전자군의 클로닝과 일시적인 유전자 발현 시스템에의 이용
남궁욱,김용주,이경광,한문희,김지영 ( Uk Nam Gung,Yong Joo Kim,Kyung Kwang Lee,Moon H . Han,Ji Young Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.3
The structural sequences of human growth hormone(hGH), human chorionic somatomammotropin(hCS)-A, hCS-B genes were isolated from a partial human genomic DNA library of pBR322. The cloned genes were identified by the restriction and Southern blot hybridization analysis. A block of highly repeated sequences was contained in the 3`-flanking sequences of hGH gene but not in the hCS-A and hCS-B genes. The structural sequences of hGH gene was fused with mouse metallothionein-I(MT-I) promoter and the expression plasmid DNA was, introduced into mouse L cells by transfection. hGH was transiently expressed in the transfected mouse L cells under the control of the mouse MT-I promoter. The secreted hGH in the medium was steadily accumulated during at least 10 days after transfection. When the MT-I promoter was induced by cadmium, the level of hGH was increased by several folds. Our results demonstrate that the plasmid vector containing the structural portion of the growth hormone gene can be used in transient expression systems for regulation studies.
남궁욱,김용주,이경광,한문희,김지영,NamGung, Uk,Kim, Yong-Joo,Lee, Kyung-Kwang,Han, Moon-H.,Kim, Ji-Young 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.3
사람 성장홀몬(hGH) 유전자와 코리온 소마토마모트로핀-A형과 B형 유전자의 구조 부위를 유전자농축방법에 의해 제조된 pBR322 사람 유전자 라이브러리로부터 분리하였다. 클로닝된 유전자들은 제한효소 지도 및 써던 하이브리다이제이션 분석에 의해 확인되었다. 반복되는 염기서열 (Repeated sequences)의 DNA 부위가 hGH 유전자의 3' 아랫쪽에 포함되어 있으나 hCS-A와 B형 유전자에는 있지 않았다. hGH 구조 유전자를 생쥐 메탈로티오닌-I(MT-I) 프로모터에 연결하여 MT-I 프로모터/hGH 융합 유전자가 포함된 재조합 플라스미드, pMThGH를 제조하였다. 이 플라스미드를 생쥐 L 세포에 넣어주었을 때 사람 성장홀몬은 MT-I 프로모터 조절하에 발현되었다. 사람 성장홀몬은 배양액으로 분비되었으며 약 10일간 생성이 지속되었다. 카드뮴으로 MT-I 프로모터를 유도하였을때 사람 성장홀몬의 농도는 몇배 증가하였다. 본 실험 결과는 사람 성장홀몬 구조 유전자가 클로닝된 플라스미드 벡터는 유전자 발현 조절 연구를 하는데 일시적인 발현 시스템에서 이용될 수 있음을 보여주었다. The structural sequences of human growth hormone(hGH), human chorionic somatomammotropin(hCS)-A, hCS-B genes were isolated from a partial human genomic DNA library of pBR322. The cloned genes were identified by the restriction and Southern blot hybridization analysis. A block of highly repeated sequences was contained In the 3'-flanking sequences of hGH gene but not in the hCS-A and hCS-B genes. The structural sequences of hGH gene was fused with mouse metallothionein-I(MT-I)promoter and the expression plasmid DNA was. introduced into mouse L cells by transfection. hGH was transiently expressed in the transfected mouse L cells under the control of the mouse MT-I promoter. The secreted hGH in the medium was steadily accumulated during at least 10 days after transfection. When the MT-I promoter was induced by cadmium, the level of hGH was increased by several folds. Our results demonstrate that the plasmid vector containing the structural portion of the growth hormone gene can be used in transient expression systems for regulation studies.
장승익,민원기,박종훈,이철상,이경광,이영원,전무형,김명희,Jang, Seung-ik,Min, Won-gi,Park, Jong-hoon,Lee, Chul-sang,Lee, Kyung-kwang,Lee, Young-won,Jun, Moo-hyung,Kim, Myoung-hee 대한수의학회 1995 大韓獸醫學會誌 Vol.35 No.1
The human homolog of position specific element of mouse Hoxa-7 was studied using transgene. It contains a 1.1 kb human DNA (HCR)- a homolog to the intergenic region between Hoxa-7 and -9, which directs the position specific expression of Hoxa-7-, tk promoter, LacZ (${\beta}$-galactosidase) gene as a reporter, and polyadenylation signal of SV40 large T antigen. It was injected into the mice embryos, and the resulting transgenic embryos were analysed through PCR as well as genomic Southern blotting with placenta DNA. Out of 20 embryos analysed, two were transgenic. Among them, one transgenic embryo expressed transgene when stained with X-gal. The expression pattern was in analogy to that of the mouse Hoxa-7, showing spatially restricted expression pattern, Since the expression of ${\beta}$-galactosidase is regulated by the upstream human HCR sequence, it implies that the HCR is the plausible position specific regulatory element of human.
이철상(Chul Sang Lee),박흠대(Hum Dai Park),정길생(Kil Saeng Chung),이경광(Kyung Kwang Lee) 한국축산학회 1989 한국축산학회지 Vol.31 No.2
Pronuclear transplantation between ICR and Fl hybrid(C57BL×CBA) mouse eggs was achieved by using a method that combines microsurgical removal of the zygote pronuclei with the introduction of donor pronuclei by a virus-mediated cell fusion technique. The technical efficiency of this procedure composed of zona cutting, enucleation, karyoplast injection and fusion step was more than 90 % at each step. Developmental rate of these nuclear transplanted embryos to blastocyst was about 60 % and when these embryos were transferred to foster mother mice. 4 nuclear transplanted mice were born.