DEVELOPMENT OF EFFICIENT PRODUCTION SYSTEM OF TRANSGENIC MINIPIG

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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