보고서 정보
주관연구기관 |
한경대학교 산학협력단 |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2012-04 |
과제시작연도 |
2011 |
주관부처 |
농림축산식품부 Ministry of Agriculture, Food and Rural Affairs(MAFRA) |
등록번호 |
TRKO201400026553 |
과제고유번호 |
1545002622 |
사업명 |
생명산업기술개발 |
DB 구축일자 |
2014-11-29
|
DOI |
https://doi.org/10.23000/TRKO201400026553 |
초록
▼
1. 연구개발 내용
○ 실험동물로 활용이 될 수 있도록 시장에서 요구하는 실험동물 조건에 부합하는 표준 생산 (개량, 번식, 사양관리) system을 구축한다.
○ 시험 생산된 Midget 미니돼지의 실험동물 규격화를 위하여 Pilot test를 실시한다.
○ 실험동물용 Midget 미니돼지 Genetic resource bank를 운용하여 개발된 Genetic resource를 보존 및 관리한다.
2. 연구개발 범위
1.실험동물용 Midget 미니돼지의 유전자원 보존 및 관리
가. Midget 미니
1. 연구개발 내용
○ 실험동물로 활용이 될 수 있도록 시장에서 요구하는 실험동물 조건에 부합하는 표준 생산 (개량, 번식, 사양관리) system을 구축한다.
○ 시험 생산된 Midget 미니돼지의 실험동물 규격화를 위하여 Pilot test를 실시한다.
○ 실험동물용 Midget 미니돼지 Genetic resource bank를 운용하여 개발된 Genetic resource를 보존 및 관리한다.
2. 연구개발 범위
1.실험동물용 Midget 미니돼지의 유전자원 보존 및 관리
가. Midget 미니돼지 유전자원 은행 준비
나. Midget 미니돼지 유전자원 은행 운영
다. 실험동물용 Midget 미니돼지 유전자원 은행
2. 실험용 Midget 미니돼지의 개량 시스템 개발
가. 기존 미니돼지 집단에 대한 유전 특성 규명
나. Midget 미니돼지 개량 시스템 운용
다. 실험동물용 Midget 미니돼지 개량 시스템 운용
3. 실험동물용 Midget 미니돼지의 생산 표준화
가. 종돈의 선발, 교배 와 개량 라인 조성
나. Midget 미니돼지 시험 생산 및 규격화
다. 실험동물용 Midget 미니돼지의 표준 생산
Abstract
▼
1. Genetic resource conservation and management of Midget miniature pigs
Part 1.
To reserve the genetic material of minipigs provided by Medikinetics, the reproductive organs were surgically removed from a total of 26 females and 4 males. In addition, 7 embryos at approximately 30 days in preg
1. Genetic resource conservation and management of Midget miniature pigs
Part 1.
To reserve the genetic material of minipigs provided by Medikinetics, the reproductive organs were surgically removed from a total of 26 females and 4 males. In addition, 7 embryos at approximately 30 days in pregnancy were extracted. Tissues that would be used for in vitro culture were placed in 37℃ home made saline. Tissues for paraffin section were frozen in liquid nitrogen (LN2). To facilitate immonological analyses and RNA probe hybridization, solutions used for block fixation were prepared to be free of DNase and RNase. Total of 77 tissues including 26 ovaries, 20 uterus, 20 oviducts, 4 testis and 7 embryos were Tissue sections were embedded in paraffin blocks.
Part 2.
In this study, we determined the full-length nucleotide sequence of MAP1LC3A (LC3A) cDNA from porcine ovary. Mixed-base oligonucleotide primers were designed based on previously cloned LC3A. The open reading frame of porcine LC3A cDNA consists of 980 bp (encoding 121 amino acids). Based on homology to the human gene (98%), this novel cDNA was identified as porcine MAP1LC3A and submitted to GenBank (Accession No. GU272221). The MAP1LC3A gene contains 4 exons and 3 introns. To map the promoter region and to investigate the presence of cis-regulatory elements, we cloned the 1051-bp fragment upstream to the transcription start site. We identified 3 TATA box and 4 CAAT box sequences in this region. There also was 23 CpG dinucleotides as potential methylation sites within this 1051-bp region. The luciferase reporter assay demonstrated that transcription factors CRE-BP, HSF, and ADR1 play pivotal roles in the expression of porcine MAP1LC3A. Indirect immuno-fluorescence with the MAP1LC3A fusion protein showed that the subcellular localization of porcine MAP1LC3A and ATG5 exhibit a punctate pattern in the cytoplasm of porcine follicular cells under stress conditions. These results indicate that MAP1LC3A can be used as an autophagosomal marker of pig follicular cell. We propose that autophagy plays a role in the maintenance of follicular development at least partially by regulating the remodeling system in porcine follicular cells.
Part 3
The objective of this study is to estimate the effect of adding TES to LEY and FGE freezing extender for the sperm viability, acrosomal morphology and DNA fragmentation from miniature pig sperm, we evaluated sperm characteristics in TFGE, TLE and LEY with various thawing condition (37℃ for 20 sec, 45 sec and 75℃ for 5 sec, respectively), and in different concentration of glycerol at 1%, 1.5%, 3%. The sperm viability and normal acrosome intact(NAI) in TFGE(Viability : 60.3±2.4, NAI : 58.6±2.2%), TLE(61.3±2.4, 62.2±2.2%) extender significantly(p<0.05) increased than that in LEY(50.2±2.4, 54.5±2.2%) extender thawed at 75℃ for 5sec. According to the results from glycerol concentration, the viability and NAI of miniature pig sperm in 1.5% glycerol TLE(66.1±3.2, 66.2±1.0%) was highest among the experimental groups. In accordance with this, DNA fragmentation rates was the lowest in TLE(43.3±0.5%) while that in LEY(63.5±2.3%) is the highest. Therefore, these results suggest that TLE extender method for freezing-thawing of miniature pig sperm increased the viability after thawing.
Part 4.
The objective of this study is to analyzed the effect of BIVM and PIVM invitro maturation medium for the oocyte maturation, Cumulus oophorus and extrusion rates of 1st polar body(pb) from the midget miniature pigs oocytes. And estimation of effect in the hormones(Adding hormones(FSH, LH and FSH+LH to PIVM) to oocyte maturation. The oocytes cumulus oophorus and extrusion rates of 1st pb in PIVM(Cumulus Oophorus : 77.1±1.4% , Extrusion rates of 1st pb : 75.3±1.6%), medium highest increased than that in BIVM(75.2±2.3% , 72.5±1.8%). According to the results from holmones effect, the oocytes cumulus oophorus and extrusion rates of 1st pb of Midget miniature pig oocytes in FSH+LH medium(PIVM+FSH+LH) was highest among the experimental groups. Also we used maturation method for oocytes invitro maturation in Midget miniature pigs, after stored freezing in LN2 box. and removed the cumulus cell from maturation oocytes, and freezing was the stored in LN2 box to the oocytes load in 0.25mm straw. Therefore, these results suggest the PIVMFL(PIVM+FSH+LH) invitro maturation medium for oocytes maturation of Midget miniature pig oocytes increased the maturation.
Part 5.
The main purpose of this study is to estimate the effect of adding Tea-N-Tris to the freezing buffer for minipig sperm. In particular, we attempted to identify the association between the MMPs expression and the survival and viability of sperms. Prior to freezing, sperms in LEY without Tea-N-Tris showed 40.3± 2.8% viability and 60.3±1.3% survival rate at 4oC. After freezing, sperms stored in LEY with Tea-N-Tris (=TLE) showed the highest viability (57.4±1.8%) and survival rate (65.6±4.6%). In accordance with this, DNA fragmentation was the highest among sperms frozen in LEY while the lowest fragmentation was observed among sperms frozen in TLE. When these sperms were used for in vitro fertilization (IVF), the LEY group showed lower rate of blastocyst development, although the difference was not statistically significant. Meanwhile the rate of blastocyst development appeared similar when sperms from TLE and TFGE(Tea-N-Tris+Fructose+Glucose+Egg yolk) group were used for IVF. We observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of MMP-9 and MMP-2 was the highest in sperms frozen in LEY, Meanwhile, sperms from the TFGE and TLE group showed lower level of MMP-9 and MMP-2 expression in the order of TLE being the lowest. Together, these results indicate that adding Tea-N-Tris to the sperm freezing buffer would not only suppress MMPs expression but also minimize DNA fragmentation, providing a mean to improve the success rate in the in vitro manipulation of minipig sperms.
Part 6.
Matrix metalloproteinases(MMP) play important roles in extracellular matrix (ECM) remodeling during ovarian follicular development, oocytes development and ovulation. In an attempt to investigate the effect of MMP activation in development cumulus-oocytes complexes, we examined the localization and expression of MMP, and monitored MMP expression profile. Cumulus-oocytes complexes were collected and matured in vitro for 24hr, 36hr and 48hr. A mRNA expression of MMP-2, MMP-9, TIMP-2 and TIMP-3 was detected in all culture medium regardless of CC, OC and COCs. Activity of MMP-2 in the OC progressively was increased from 24hr to 48hr. But MMP-9 was not detected in all culture medium. The localization of MMP-2 was also measured by immunohistochemistry analysis. The MMP-2 and TIMP-2 was detected in cumulus cell and oocyte zona pellucida. Expression of MMP-2 protein in the COCs was progressively increased from 24hr to 48hr. However, MMP-9 protein was progressively decreased from 24hr to 48hr. And TIMP-2 protein was most highly expressed in the COCs 36hr. Expression of TIMP-3 protein in the COCs was progressively increased from 24hr to 48hr. In conclusion, these results suggest that MMP-2 plays a role in maintaining normal maturation and development by controlling the ECM inhibitor concentration on cumulus cell and oocytes.
2. Development of improved systems of Midget miniature pigs
This study was conducted to estimate the growth curve parameters for the body weight (BW) and the body length (BL) of miniature pig in Korea. Growth curve parameters were estimated through anonlinear regression modelusing Gompertz, Logistic and von Bertalanffy methods. Atotal of 25 piglets were measured seven times with two months interval to estimate both body weight and length. Results showed that the estimated average values for the body weight (body length) were 31.83 kg (58.77) for the mature weight(A), 3.06 (1.74) for the growth ratio (β), and 0.28 (0.52) for the maturing rate(κ). Average inflection points showing maximum growth rate estimated each month for body weight were 3.97 kg and 11.70 cm, while for the body length were 1.06 kg and 21.61 cm. Moreover, the estimated maturation rates of the body weight and length for the Group of Sire 1 were respectively about 0.22 and 0.40, whereas for the Group of Sire 2 these values were 0.34 and 0.39. On the other hand, for the Groups of Dam 1, Dam 2, and Dam 3 maturation rates for their body weights were respectively about 0.26, 0.28 and 0.33, while for their body lengths these values were 0.43, 0.37 and 0.38. The study also indicated a negative relationship between the values of mature weight and maturity rate for the body weight will result to a higher inflection point which is in contrast for the body length where results show that a positive relationship between the values of mature length and the maturity rate will result to a higher inflection point.
Furthermore, the growth performance of miniature pig varies across stages but using these estimated growth curve parameters could improve the genetic traits of miniature pig.
3. Standardize the production of Midget miniature pigs
This study was conducted to develop the midget minipig from the Micropig herds for pharmacological studies and nonclinicla trials. To downsize minipig, we selected tiny minipigs and mated them. After births, the body weight and body length of offspring were measured periodically and estimated. The midget minipig herds were composed of 5 strains that were two sire's lines and three dam's lines, To improve the downsing rate, some lines were mated among the midget minipig herds and produced genetically normal offsprings. From 2011, Medikinetics has customized the midget minipigs as laboratory animals for biomedical researches. By genetic analysis of hair coat color and size, some data for the status and breeding plan of micropig herds were acquired and the customized production system was developed
목차 Contents
- 표지 ... 1
- 제출문 ... 2
- 요약문 ... 3
- SUMMARY ... 10
- CONTENTS ... 15
- 목 차 ... 17
- 제 1 장 서 론 ... 19
- 제 1 절 연구개발의 목적 ... 19
- 제 2 절 연구의 필요성 ... 19
- 제 3 절 활용방안 및 기대성과 ... 20
- 제 2 장 국내외 기술개발 현황 ... 22
- 제 1 절 국내 제품생산 및 시장 현황 ... 22
- 제 2 절 국외 관련(유사)제품의 생산 및 시장 현황 ... 22
- 제 3 장 연구개발수행 내용 및 결과 ... 23
- 제 1 절 실험동물용 Midget 미니돼지의 유전자원 보존 및 관리 ... 23
- 제 2 절 실험동물용 Midget 미니돼지의 개량 시스템 개발 ... 49
- 제 3 절 종돈 선발, 교배와 개량 라인 조성 ... 67
- 제 4 장 연구개발목표 달성도 및 대외기여도 ... 71
- 제 1 절 목표대비 대외달성도 ... 71
- 제 2 절 목표대비 연구결과 ... 71
- 제 3 절 정량적 성과(논문게재, 특허출원, 기타)를 기술 ... 72
- 제 5 장 연구개발결과의 활용계획 ... 74
- 제 1 절 연구 개발결과 ... 74
- 제 2 절 연구 개발결과 활용계획 ... 75
- 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 75
- 제 7 장 참고문헌 ... 76
- 끝페이지 ... 80
※ AI-Helper는 부적절한 답변을 할 수 있습니다.