초록 ▼

1차년도(1998)
연구개발목표
- 수핵난자의 확보와 미세조작
- Somatic cell line 수립 및 배양조건 확립
- 유·사산관련 virus성 질병검색
- 미성숙란의 동결보존 기술개발
연구개발 내용 및 범위
·고능력 특정질병 free 젖소 선정
·도축장 유래 미성숙 난포란의 적정 체외성숙배양 조건 설정
·Somatic cell line 수립
·수핵난자의 탈핵기법 확립
·모축우군의 질병 발생 검색
·성숙란의 동결보존 방법의 개선
2차년

1차년도(1998)
연구개발목표
- 수핵난자의 확보와 미세조작
- Somatic cell line 수립 및 배양조건 확립
- 유·사산관련 virus성 질병검색
- 미성숙란의 동결보존 기술개발
연구개발 내용 및 범위
·고능력 특정질병 free 젖소 선정
·도축장 유래 미성숙 난포란의 적정 체외성숙배양 조건 설정
·Somatic cell line 수립
·수핵난자의 탈핵기법 확립
·모축우군의 질병 발생 검색
·성숙란의 동결보존 방법의 개선
2차년도(1999)
연구개발목표
- 수핵난자의 세포질 활성화 기술확립
- 핵이식난의 체외배양 및 이식
- 특정질병 free somatic cell line 공급
- Somatic cell의 핵이식을 위한 신개념의 동결법 개발
연구개발 내용 및 범위
·세포질 활성화 조건 설정
·somatic cell과 수핵난자의 융합조건 설정
·신속 정밀 항원검출법(PCR)개발 및 효능 분석
·상이한 발육단계의 미성숙난자의 동결등의 검토
3차년도(2000)
연구개발 목표
- 우량소 복제란의 대량생산을 위한 체외배양체계 및 동결보존법 개발(시험이식 병행)
- GFP 유전자 도입 somatic cell line 작성
- GFP 유전자 도입 somatic cell 유래 복제 수정란의 생산 및 이식
- 인간 α1-AT) 유전자의 소 체세포로의 안정적 도입
- 인간 erythropoietin(EPO) 유전자의 조직 특이 발현 vector 개발
연구개발 내용 및 범위
- 소 복제수정란의 최적 체외배양체계 설정
- 우량소 복제란 대량생산 및 이식
- 유전자 도입 최적조건 설정
- GFP 유전자 도입 복제 수정란의 체외배양 조건 확립
- 시험이식 및 수태 검정
- 인간 α1-AT 유전자 발현 vector 제조
- 유전자 안정적 도입 방법 개발
- 소의 유선조직 특이 발현 vector 개발
- 인간 EPO 유전자의 유선조직 특이 발현 vector 제조
- EPO 발현 vector의 유선세포 발현여부 검증

Abstract ▼

Due to the decline in the livestock industry in Korea, a research was financed by a grant from the ministry of Science and Technology, "Cloning of high-performance cows by somatic cell nuclear transplantation". The mission was to develop a system enabling the mass production of high performance cows

Due to the decline in the livestock industry in Korea, a research was financed by a grant from the ministry of Science and Technology, "Cloning of high-performance cows by somatic cell nuclear transplantation". The mission was to develop a system enabling the mass production of high performance cows by somatic cell nuclear transfer within a project duration date of 4 years.
First year research objectives were 1) Prepare recipient oocytes and perform micro manipulation, 2) Establish somatic cell line, 3) Determine the diseases which cause embryonic death and abortion, and 4) Cryopreserve of immature oocytes, Our achieved maturation and enucleation rates were repectively 73 % and 95%. Moreover, we established somatic cell lines collected from 8 high-performance Holsteins and 7 Hanwoo(Korean native) cows. Furthermore, we were able to pre-screen the bovine viral diarrhea virus. At which time, we developed an optimal cryopreservation protocol for immature oocytes. However, we completed our final objective in the 1st year of our research (birth of cloned Holstein [Young-long] and Hanwoo [Jeanie] calves). Therefore, our 4 year projection plan was at tered.
Second year research objectives were 1) Establish an effective protocol for activation of recipient oocytes and in vitro culture (IVC) of cloned embryos, 2) Transfer of NT embryos to the recipient cows, 3) Establish a disease free somatic cell line, and 4) Cryopreserve matured oocytes. Activation rate was achieved 90% and a cleavage rate of 93% by the chemical activation. Additionally, developmental rate to blastocyst was up to 45%. Then we transferred somatic NT embryos to 1,250 recipient cows throughout the country. We also developed the effective cryopreservation method of mature oocytes having 60% post thaw normal morphology.
Third year research objectives were 1) Establish of IVC and cryopreserve systems for mass production and supply of somatic NT embryos throughout the country, 2) Establish green fluorescent protein (GFP)-transfected somatic cell lines, 3) Produce of GFP-transfected somatic cells derived NT embryos, 4) Stable transfection of α1-antitrypsin gene to bovine somatic cell and 5) Develop tissue-specific erythropoietin (EPO) expression vector. We achieved satisfactory developmental rates of NT embryos in medium having 1.5-3. 0mM of fructose. Then, we supplied 1,250 embryos to Ministry of Agriculture and Forestry, and transferred embryos to 250 recipient cows in experimental farms. When different gene transfection method were compared, liposome-mediated methods were more effective than that of electroporation and Ca phosphate. Nevertheless, we achieved 70% developmental rate of GFP-transfected embryos and transferred transgenic embryos to the recipient cows. We also performed stable transfection of pEGFP-BLA-AT85 ( plasmid-promoter-target gene) by liposome-mediated method and we developed 2 vector which were expressed in mammary gland.
Our research findings show mass production of high-performance cows using somaticcell NT is possible for the rapid development in the livestock industry. Since we achieved our final aim in the 1st year of our research, we partially changed purpose of this research to production of transgenic cows. And then we proposed early completion of this research for an additional research.

목차 Contents

• 제 1장 서론...15
• 제 2장 국내외 기술개발 현황...17
• 제 3장 연구개발수행 내용 및 결과...19
• < 제 1차년도 >...19
• < 제 2차년도 >...35
• < 제 3차년도 >...43
• 제 4장 연구개발목표 달성도 및 대외기여도...63
• 제 5장 연구개발결과의 활용계획...64
• 제 6장 참고문헌...65