DEVELOPMENT OF EFFICIENT PRODUCTION SYSTEM OF TRANSGENIC MINIPIG

转基因小型猪高效生产体系的开发

• 批准号: 11358015
• 负责人: SAKUMA Ichiro
• 金额: $ 18.56万
• 依托单位: THE UNIVERSITY OF TOKYO
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11358015/
• 关键词: transgenic mini pig; con-focal laser scanning microscope; gene transfer; micro porous glass; micro manipulator; enhanced green fluorescent protein; 遺伝子組みえ替えミニブタ; 三次元視硬性内視鏡; ミニブタ卵胞卵; レーザ共焦点顕微鏡; 水浸型対物レンズ; 遺伝子組み換えミニブタ; 培養卵保持MPGマイクロポケット; 低侵襲採卵

Development of the efficient system for creating transgenic miniature pig has a strong impact on supply of cells, tissue, and organs for xeno-transplantation. Although the production of transgenic offspring in the miniature pig have been demonstrated by the transfer of microinjeced in-vivo generated embryos, this approach suffers from the major drawback that the experimental turnover is long and expensive, and only relatively few offspring can be obtained for any particular treatment. Successful in-vitro methods for embryo production, micromanipulation and the selection of transgenic embryo prior to embryo transfer could improve the efficiency of the production of transgenic offspring. This study was carried out to investigate the effect of sexual maturation of pig on embryo production in-vitro. the effect of timing of gene injection to pronucleus on the development of pig embryo and to examine the effect of enhanced green fluorescence protein (EGFP) gene injection as a reporter on the … More selection of transgenic pig embryo. On the other hand, we developed ova culture system where ova are fixed and cultured in small pockets on MPG (micro porous glass) under the flow of culture medium and the process of culture can be continuously monitored by a confocal laser-scanning microscope. Introduction of a confocal microscope enables measurement of three-dimensional shape of ova during culture. We also studied the automation system for gene injection. The results of the study are as follows ; 1) we could culture ova by the newly developed flow culture system, 2) the efficient production of in-vitro generated embryo is achieved by using ova from sexually matured sow, 3) it is possible to inject transgene to the pig ova 12 hours after in-vitro fertilization, 4) efficiency of EGFP gene expression is influenced by types of promoter and length of DNA fragment, 5) use of EGFP gene as a marker for embryo selection is suitable for pig embryo at 4-cell and blastocyst stages, and 6) it is effective to use automatic apparatus in gene injection under microscope to improve the efficiency of production of transgenic embryo of miniature pigs. Less

建立高效的转基因小型猪系统对异种移植的细胞、组织和器官的供应具有重要影响。虽然小型猪的转基因后代是通过移植显微注射体内产生的胚胎来证明的，但这种方法的主要缺点是实验周转时间长且成本高，并且对于任何特定的处理只能获得相对较少的后代。成功的胚胎生产、显微操作和胚胎移植前选择转基因胚胎的体外方法可以提高转基因后代的生产效率。本研究旨在探讨猪性成熟对体外胚胎生产的影响。原核基因注射时机对猪胚胎发育的影响及增强型绿色荧光蛋白基因注射对…的影响更多转基因猪胚胎的选择。另一方面，我们开发了卵子培养系统，该系统将卵子固定在微孔玻璃上，在培养液流动的情况下，将卵子固定在小袋中进行培养，并通过激光共聚焦扫描显微镜连续监测培养过程。共聚焦显微镜的引入使人们能够在培养过程中测量卵子的三维形状。我们还研究了基因注射的自动化系统。研究结果如下：1)利用新开发的流式培养系统可以进行卵子的培养；2)利用性成熟母猪的卵子进行体外胚胎的高效生产；3)可以在体外受精12小时后将转基因注射到猪的卵子中；4)绿色荧光蛋白基因的表达效率受启动子类型和DNA片段长度的影响；5)以绿色荧光蛋白基因作为胚胎选择的标记适合于4-细胞期和囊胚期的猪胚胎；6)在显微镜下使用自动基因注射装置可以有效地提高小型猪转基因胚胎的生产效率。较少

项目成果

Yoshida,M: "Conservation of sperms : current status and new trends."Anim.Reprod.Sci.,. 60. 349-355 (2000)

吉田，M：“精子的保护：现状和新趋势。”Anim.Reprod.Sci.，。

辻隆之,阿部茂,小倉敬行,蔭山雅敏,辻敬之,佐久間一郎,土肥健純,吉田光敏,大久津昌治: "遺伝子組み換えミニブタ作出用卵胞卵培養環境システムの開発"医用電子と生体工学. 38(特別号). 410 (2000)

Takayuki Tsuji、Shigeru Abe、Takayuki Ogura、Masatoshi Kageyama、Noriyuki Tsuji、Ichiro Sakuma、Kenzumi Doi、Mitsutoshi Yoshida、Masaharu Okutsu：“用于生产转基因迷你猪的卵泡卵培养环境系统的开发”医学电子与生物工程38（专题。问题）410（2000）。

Yoshihiko NAKANISHI: "A Challenge to xenotransplantation-From transgenic minipigs to creation of mammoth"Experimental Medicine. 17. 36-41 (1999)

中西义彦：“异种移植的挑战——从转基因小型猪到猛犸象的创造”实验医学。

中西喜彦: "異種移植にかける夢"実験医学. 17. 36-41 (1999)

Yoshihiko Nakanishi：“异种移植之梦”实验医学 17. 36-41 (1999)。

大谷直人,伊藤理恵,田中保積,吉田光敏,辻隆之,中西喜彦: "電気融合法を用いたミニブタ体細胞核移植における胚盤胞の作出"第93回日本繁殖生物学会大会講演要旨. 74 (2000)

Naoto Otani、Rie Ito、Yasumi Tanaka、Mitsutoshi Yoshida、Takayuki Tsuji、Yoshihiko Nakanishi：“使用电融合法在小型猪体细胞核移植中创建囊胚”日本生殖生物学学会第 93 届年会摘要 74（2000 年）。 ）