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동해방지제 , 식빙 , 동결속도 및 보존기간이 생쥐 초기배의 생존성에 미치는 영향
진동일,임경순,오봉국,이용빈 ( D . I . Jin,K . S . Im,B . K . Ohh,Y . B . Lee ) 한국축산학회 1986 한국축산학회지 Vol.28 No.7
To study the factors affecting cryopreservation of mouse embryos, 6-12 week old ICR mice were used. Embryos were dehydrated in IPBS medium containing DMSO or Glycerol by 3 step procedure and cooled to -50℃ at different cooling rates (1.0, 3.0, 5.0, 7.0 and 10.0℃/min) and plunged into liquid nitrogen (-196℃) and stored for 12hrs to 30 days. Embryos were thawed in air to room temperature at rate of about 20℃/min and rehydrated by 3 step procedure. When the embryos were dehydrated in the medium containing cryoprotectants, then survival rates were decreased compared to embryos which did not dehydrated, and same trend was noted when the ice nucaeation of embryos was induced at -5℃. The survival rate of embryos dehydrated or cooled to -5℃ showed no difference between 8-cell and morula stage embryos. The survival rate of embryos was best (45%) when the cooling rate was 1℃/min, but it was decreased when the cooling rate was increased. Embryos could be stored up for 15-30 days without significant decrease in their survival rate when they were cooled at 1℃/min to -50℃ and stored in -196℃.
토끼에서 반복적인 과배란유도가 배란율과 난자의 체외 발육율에 미치는 영향
진동일,임경순,이홍미 韓國受精卵移植學會 1997 한국동물생명공학회지 Vol.12 No.3
토끼에서는 수정란 이식과 같은 기본적인 번식공학적 방법의 효율성이 아직 생쥐와 같은 실험동물에 비해 떨어지고 있어 생물공학적인 기술을 응용하는데 큰 어려움이 있다. 특히 유전자 이식에 의한 형질전환 토끼의 생산과 같은 생물공학적인 기술을 실용화하는데 효율이 높은 수정란이식 기술의 개발이 필수적이라고 할 수 있다. 본 연구에서는 토끼에서 수정란 이식 기술의 첫 단계인 과배란 유도를 효율적으로 이용할 수 있는 방법을 정립하기 위해 반복적인 과배란 유도가 배
Effects of Slow Freezing on Development of Blastomeres Separated from Mouse Preimplantation Embryos
진동일 한국가축번식학회 2000 Reproductive & developmental biology Vol.24 No.3
The development of single blastomeres isolated from 2-, 4- and 8-cell mouse embryos and the ability of such blastomeres to survive slow freezing were studied. Of 223, 60 and 188 single blastomeres isolated from 2-, 4- and 8-cell mouse embryos, respectively, 111 blastomeres (49.8%) from 2-cell embryos, 12 blastomeres (20.0%) from 4-cell embryos and blastomeres (16.5) from 8-cell embryos developed into blastocysts after culture for 96 hrs. The recovery rate was 54.2% (65/120), 46.4% (13/28) and 24.3% (17/70) of blastomeres derived from 2-, 4- and 8-cell embryos following freezing and thawing and the survival of frozen-thawed blastomeres was 27.1% (16/59), 36.4% (4/11) and 17.6% (3/17), and respectively. The apparently six normal fetuses were obtained from frozen-thawed blastomere from 2-cell embryos after transferring into the recipients. These results indicate that mouse blastomeres isolated from preimplatation stage embryos can survive storage in liquid nitrogen following slow freezing.
진동일 한국수정란이식학회 2000 한국동물생명공학회지 Vol.15 No.2
Transgenic rabbits were produced by DNA microinjection using growth hormone receptor (GHR) and IGF-1 receptor (IGF-1R) genes. Overall efficiencies for production of transgenic rabbits were 3.2% and 3.1% in GHR and IGF-1R genes, respectively. Founder rabbits transmitted transgenes to their progenies through medelian fashion. Growth rate in GHR and IGF-1R transgenic rabbits was faster than non-transgenic rabbits. Transgenic rabbits grew larger (25% and 15% increase in body weight of GHR and IGF-1R transgenic rabbits, respectively) than non-transgenic rabbits and organ weight of transgenic rabbits increased, suggesting that GHR and IGF-1 genes affects growth rates in transgenic rabbits.
Tetraploidy Induction of Mouse Embryos by In Vitro Culture with Cytochalasin B
진동일 한국수정란이식학회 1999 한국동물생명공학회지 Vol.14 No.2
효율적인 homozygous동물을 생산하기 위한 실험의 단계로 염색체가 4배체인 수정란의 이용성을 타진하기 위해 생쥐 수정란과 cytochalasin B를 사용하여 4배체 유도에 관한 실험을 수행하였다. 생쥐 2-세포기 수정란을 10 ㎍/ml 농도의 cytochalasin B로 약 20시간 배양하였을 때 모든 수정란은 발육을 거의 멈추었으나, 이 수정란을 cytochalasin B-free medium에 체외배양 하였을 때 발육이 재개되어 48시간 후 상실기나 배반포기까지 약 74%의 발육율을 나타내었다. 그러나 발육된 수정란의 세포수는 대조구에 비해 훨씬 적은 것으로 나타났다. 염색체 분석결과 cytochalasin B로 처리한 대부분의 수정란은 4배체인 것으로 나타났고 약간의 수정란은 mosaicism과 다배체를 나타내기도 하였다. 그러므로 cytochalasin B를 이용하여 효과적으로 4배체의 수정란을 유도할 수 있는 것으로 나타났다.
동결과정중 임계온도의 추정과 동결 융해한 생쥐 초기배의 이식후 생존성
진동일,임경순,오봉국,이용빈,정진관,김희석 ( D . I . Jin,K . S . Im,B . K . Ohh,Y . B . Lee,J . K . Jung,H . S . Kim ) 한국축산학회 1986 한국축산학회지 Vol.28 No.11
Six to twelve weeks old ICR mice were used as besic study on cryopreservation of mouse embryo to estimate critical temperatures of mouse embryo during freezing, and to study survival rate of frozen mouse embryo transfered to recipients. Embryos cooled at 1℃/min to -50℃ or -60℃ before plunging into liquid nitrogen had best survival rates. When the embryos were cooled at 7℃/min to -10℃, 60% of embryos was survived, but the survival ;rate was decreased when temperature at plunging into liquid nitrogen was lowered. Generally morula stage embryo was more tolerable against cooling than 8-cell stage embryo. The survival rate of embryos which were cooled to -40 or -50℃ at rate of 1 or 7℃ per min. and plunged into LN₂ was higher in morula than in 8-cell embryos exposed both in DMSO and glycerol. When the embryos were transferred into recipients, implantation rate was 12.2% in frozen embryo and 23.8% in fresh embryo.
진동일 선문대학교 ·중소기업기술지원연구소 1999 선문공대 연구/기술 논문집 Vol.4 No.1
토끼 배아세포(Embryonic Stem Cell)를 분리하기 위해 토끼 1-cell embryo를 채란하여 in vitro에서 blastocyst까지 배양한 후 mouse embryonic fibroblasts(MEF), rabbit embryonic fibroblasts(REF) 및 STO cell expressing Leukemia Inhibition Factor gene(SNL) feeder cell과 공배양하였다. 외관상 충실한 토끼 배아세포 8 개를 확보하였고 분리된 토끼 ES ceIl의 모양은 주위에 분화된 세포가 없는 전형적인 colony모양으로 성장하고 액체질소에 동결보존 및 3-5차례의 계대배양 후에도 이러한 모양은 계속 유지되었다 충분히 자란 dish를 1 : 2로 계대배양을 한 후 다시 confluent하게 자라는 데에 걸리는 시간(doubling time)은 빠른 경우 84시간으로 나타났다. 분리된 토끼 ES cell은 gelatin이 coating되지 않은 culture dish에 이식 배양하였을 때 부유상태로 증식하면서 내부에 강(腔)이 생기고 외배엽과 내배엽이 형성하는 전형적인 Embryoid body 모양을 나타내어 분리된 ES cell이 미분화상태의 stem cell임이 확인되었다. To establish rabbit Embryonic Stem (ES) cells, rabbit one-cell embryos were collected and cultured in vitro to blastocysts. Blastocysts were co-cultured with mouse embryonic fibroblasts (MEF), rabbit embryonic fibroblasts (REF) or STO cells expressing LIF (SNL). Although rabbit ES cells were isolated with low efficiencies, total 8 ES cell lines were kept in vitro with normal colony shape. Their doubling time was about 84 hours and their shapes were maintained with passing and freezing. These rabbit ES cells were differentiated into embryoid body following the culture in the uncoated dishes which indicated that they were undifferentiated stem cells.