초록

○ 연구결과
- 사람 tPA 유전자 및 유산양 β-casein프로모터 클로닝
- 형질전환생쥐의 생산 및 유즙에서 사람 tPA 단백질 발현분석
- 재란산양(흑염소)난소의 이용기술의 확립 및 핵이식 수정란의 이식
- 핵이식란의 이식 후 복제산양생산 형질전환수정란 이식
- 1 마리 복제산양 생산

Abstract ▼

IV. Research Results
To establish efficient somatic nuclear transfer technology for dairy goat, construction of transgenes, transfection into goat fetal fibroblast cells, production of reconstituted embryos and transfer of reconstituted embryos into recipients were performed in this study. Finall

IV. Research Results
To establish efficient somatic nuclear transfer technology for dairy goat, construction of transgenes, transfection into goat fetal fibroblast cells, production of reconstituted embryos and transfer of reconstituted embryos into recipients were performed in this study. Finally one coned dairy goats was produced using Korean native goat oocyte with dairy goat fetal fibroblast cells. Results of this study are as follows;
1. Transgene and expression vectors for dairy goat
1) Cloning of human tPA gene from human fetal lung cell lines by RT-PCR
Human tPA cDNA was cloned by RT-PCR with mRNA of human fetal lung cell line.
2) Cloning of goat β-casein promoter sequence
Goat β-casein promoter sequence (3 kb) was cloned by PCR with Saanen genomic DNA and confirmed by sequencing.
3) Cloning of 5' and 3' flanking sequences of goat β-casein promoter region for knock-in vector construction
Primers deduced from previously reported goat β-casein promoter sequence and exon 1 sequence were used for cloning of 5'flanking region and primers from exon 3 and exon 7 were used for cloning of 3' flanking region. PCR with genomic DNA was used for clonging these flanking regions.
4) Construction of trangene for transgenic goat
Firstly to analyze integration expression of foreing gene in cell and embryo, pCX-EGFP was linked with Neor gene. Secondly human tPA cDNA was ligated to pBC1(goat β-casein promoter) for the transgenic mouse and goat production. Thirdly using 5' promoter flanking DNA and 3' exon flanking DNA, knock-in vector was constructed with tPA cDNA.
5) Establishment of goat fetal fibroblast cell lines
Saanen fetal fibroblast cells were established using day 50 fetuses, sexed by PCR and stored in liqid nitrogen.
2. Establishment of IVM and IVF of goat oocytes
1) Establishment of IVM of Korean native goat oocytes
Korean native goat ovaries delevered from slaughter house were used for oocyte source of nuclear transfer of dairy goat cells. For IVM of Korean native goat oocytes. TCM199 containing 10% FBS, 5 ug/ml FSH, 10 ug/ml LH and 10 ug/ml estrogen was used.
2) Establishment of IVF of Korean native goat oocytes
Matued oocytes were fertilized with capacitated goat sperm and then cultured in vitro. About 50% of fertilzed embryos developed to morula/balstocyst stage.
4) Freezing storage of goat semen for the IVF
For continuous supply of goat semen, good quality of semen in fall season were collected and frozen.
3. Establishment of efficient nuclear transfer methods in dairy goat
1) Establishment of activation methods for goat oocytes
Activation condition was established by performing experiments of parthenogenetic development.
2) Establishment of fusion condition for reconstituted embryo
Fusion condition and donor cell cycle were tested following reconstituted embryos.
3) Analysis of GFP expression in reconstituted embryos
pCX-EGFP gene were transfected into fetal firobalst cells and fetal fibroblast cells expressing GFP were selected. Subsequently those cells were used for nuclear transfer. Reconstituted embryos were analyzed for the expression of GFP.
4. Production of trangenic mice containing human tPA gene
1) Production and analysis of trangenic mice containing human tPA gene Transgenic mice (5 lines) were produced by microinjection of β-casein promoter-tPA transgene into zygotes. F₁ and F₂ female transgenic mice were bred to obtein milk following parturition.
2) Analysis of trangenic mice
Western blot analysis was carried out to detect secretion of human tPA protein in the transgenic milk using anti-tPA antibody. Milligram level of human tPA protein per one liter milk was secreted from transgenic mammary gland.
5. Transfection of transgene into fetal fibroblast cells
1) Transfection of EGFP gene into fetal fibroblast cells
pCX-EGFP-Neor gene was transfected into goat fetal fibroblast cells and neomyci selection was carried out to select cells containing pCX-EGFP-Neor gene
2) Transfection of EGFP gene into fetal fibroblast cells
β-caseinpromoter-human tPA cDNA transgene and PGC-Neor gene were co-transfected into goat fetal fibroblast cells and cells containing transgene was selected through neomycin resistance.
6. Development of efficient embryo transfer methods for transgenic cloned embryos
1) Establishment of synchronization method for goat recipients
For the synchronization of recipients, CIDR and PGF2α-treatment experiment was performed to establish appropriate synchronization method.
2) Analysis of estrus and ovulation in synchronized recipients
Following synchronization, recipients were analyzed to examine occurrence of estrus and ovulation.
7. Production of cloned and transgenic dairy goats
1) Establishment of in vitro culture conditions for the reconstituted embryos
Following nuclear transfer of fetal fibroblast cells into Korean native goat oocytes, development of reconstituted embryos in vitro was evaluated. About 20% of those embryos developed upto morula/blastocyst stage.
2) Analysis of tansgene integration in reconstituted embryos
Following nuclear transfer of fetal fibroblast cells containing EGFP gene and tPA gene into oocytes, reconstituted embryos was analyzed to detect transgene integration.
3) Transfer of reconstituted embryos into oviducts of recipients
Reconstituted embryos were surgically transferred into oviducts of synchronized recipients. Following transfer, ultrasound-pregnancy test was carried out at day 50.
4) Production of cloned dairy goat
One cloned fetus was successfully produced by nuclear transfer of dairy goat fetal fibroblast cells into Korean native goat oocytes and embryo transfer into CIDR-induced recipient.
5) Analysis of genotype of cloned goat
Genotype of cloned goat was analyzed by PCR single stranded polymorphism of goat major histocompatibility complex class II DRB gene locus. The genotype of cloned goat was matched to original fetal fibroblast cell.
6) Transfer of reconstituted embryos with cells containing β-caseinpromoter-human tPA cDNA transgene into synchronized recipients
A total of 422 reconstituted embryos containing β-caseinpromoter-human tPA transgene were transferred into oviducts of 25 synchronized recipients.
Eleven recipients were identified not to be pregnant and nine recipients were not delivered. Others are waiting to term.

목차 Contents

• 표지 ... 1
• 제출문 ... 3
• 요약문 ... 5
• SUMMARY ... 11
• CONTENTS ... 21
• 목차 ... 23
• 제1장 연구개발과제의 개요 ... 25
• 제1절 연구개발의 필요성 ... 25
• 1. 기술적 측면 ... 25
• 2. 경제·산업적 측면 ... 29
• 3. 사회·문화적 측면 ... 32
• 제2절 연구개발 내용 및 범위 ... 34
• 1. 연구개발 목표와 내용 ... 34
• 2. 연차별 연구개발 목표와 내용 ... 35
• 제2장 국내외 기술개발 현황 ... 40
• 제1절 국내외 관련기술의 현황 ... 40
• 제2절 앞으로의 전망 ... 43
• 제3장 연구개발수행 내용 및 결과 ... 44
• 제1절 서론 ... 44
• 제2절 연구재료 및 방법 ... 46
• 제3절 연구결과 및 고찰 ... 53
• 제4절 결론 ... 88
• 제4장 목표달성도 및 관련분야에의 기여도 ... 90
• 1. 유산양용 유전자 벡터의 구축 (달성도: 100%) ... 90
• 2. 유산양 난자제조 기술의 확립 (달성도: 100%) ... 91
• 3. 형질전환생쥐의 생산 및 유전자 도입 수정란의 생산 (100%) ... 93
• 4. 핵이식란 및 형질전환수정란의 이식 (100%) ... 93
• 5. 형질전환유산양 및 형질전환생쥐의 유전자 분석 (100%) ... 94
• 6. 복제 및 형질전환 유산양의 생산 및 관리 (100%) ... 95
• 제5장 연구개발결과의 활용계획 ... 96
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 97
• 제7장 참고문헌 ... 99
• 끝페이지 ... 102