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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.
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.
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.
형질전환 생쥐에서 인간 혹스 조절인자에 의해 유도된 리포터 Transgene 의 역동적 발현패턴
신재승(Jae Seung Shin),박성도(Sung Do Park),권윤정(Yun Jeong Kwon),이철상(Chul Sang Lee),허만욱(Man Wook Hur),박형우(Hyoung Woo Park),김명희(Myoung Hee Kim) 한국유전학회 2001 Genes & Genomics Vol.23 No.4
In the previous experiment, we have transiently analyzed a 307 bp cis-acting regulatory element of HOXA-7 in transgenic mice (TG). Since it was sufficient to direct a reporter gene to be expressed at the specific region of day 12.5 post coitum (p.c.) embryo with a sharp anterior boundary, we generated transgenic mice line using the NM307 (307 bp/tk promoter/LacZ/SV40 poly A) construct in order to see the temporal expression pattern following embryonic development. Through polymerase chain reaction (PCR) with LacZ specific primers and genomic DNAs isolated from ear tissue, 4 transgenic mice (founder mice, F0) were obtained and 3 exhibited characteristic expression pattern. The expression detected as early as day 8.5 p.c. in the neuroectoderm and epiblast at the posterior region; from the closed neural tube around the region S10-12 to the adjacent posterior neural fold, neural groove and to the posterior epiblast along the anteroposterior axis. At day 7.5 p.c., however, LacZ expression was not detected. As embryo develops, the expression was restricted in the neural tube from S10 area to the end of tail of day 9.5 p.c. embryo, among which rather low level of expression was detected particularly in the region of S10-13, and the overall expression pattern continued through the following day. From day 11.5 to 12.5 p.c. weak spreading of the expression was detected to the anterior of S10 and a repression at the tail region. These results altogether indicate that the 307 bp is a temporal as well as a spatial regulatory element of HOXA-7 initiating the function at around day 7.5.~8.5 p.c. and continuing through mid gestation.