DEVELOPMENT OF EFFICIENT PRODUCTION SYSTEM OF TRANSGENIC MINIPIG

建立高效的转基因小型猪系统对异种移植所需的细胞、组织和器官的供应具有重要影响。尽管已经通过显微注射体内产生的胚胎的转移证明了在小型猪中产生转基因后代，但是这种方法的主要缺点是实验周转时间长且昂贵，并且对于任何特定处理只能获得相对较少的后代。成功的体外胚胎生产方法、显微操作和胚胎移植前转基因胚胎的筛选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。研究了原核基因注射时机对猪胚胎发育的影响，以及增强型绿色荧光蛋白（EGFP）基因注射对猪胚胎发育的影响。 关于我们 转基因猪胚胎的筛选。另一方面，我们开发了卵细胞培养系统，其中卵细胞在培养基流动下固定并培养在MPG（微孔玻璃）上的小口袋中，并且培养过程可以通过共聚焦激光扫描显微镜连续监测。共聚焦显微镜的引入使得能够在培养期间测量卵子的三维形状。本文还对基因注射自动化系统进行了研究。研究结果如下：1）我们可以通过新开发的流动培养系统培养卵子，2）通过使用来自性成熟母猪的卵子实现体外产生胚胎的高效生产，3）可以在体外受精12小时后将转基因注射到猪卵子中，4）增强型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）使用增强型绿色荧光蛋白基因作为胚胎选择标记适用于4细胞期和囊胚期的猪胚胎，6）显微镜下使用自动化基因注射装置可以有效提高小型猪转基因胚胎的生产效率。少

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