哺乳動物細胞における脱分化ならびに全能性獲得機構の解明

本研究旨在阐明牛体细胞脱分化或获得全能性过程中基因表达的特异性。事实上，报告基因的制备、转基因细胞系的建立、利用转基因供体细胞核移植生产重组胚胎以及重组胚胎的基因表达等方面都进行了研究。得到了以下结果。1.成功地从插入pGK启动子的绿色荧光蛋白载体中制备了报告基因。2.GFP载体在牛成纤维细胞中的表达，经筛选后2天和20天后，GFP的表达达35-55%。3.通过GFP阳性细胞的核移植获得重组胚胎。4.将GFP阳性细胞从1-细胞期移植到囊胚期，在重组牛胚胎中观察到了GFP的表达。这些结果表明，通过核移植将外源基因导入牛胚胎是可能的，并且外源基因在整个胚胎中都有表达。

The present study was carried out to clarify the specificity of gene expression during dedifferentiation or acquisition of totipotency in bovine somatic cells. In fact, the preparation of reporter gene, establishment of gene transferred cell lines, production of reconstituted embryos by nuclear transfer using gene transferred donor cells and gene expression of reconstituted embryos were investigated. The following results were obtained. 1. The preparation of reporter genes from GFP vectors inserted with PGK promorter between EcoRI and PstI were obtained successfully. 2. The GFP expression in transfected bovine fibroblast cells with GFP vector was observed 35-55% after 2 and 20 days after selection. 3. The reconstituted embryos were obtained by a nucleat transfer with GFP-positive cells. 4. GFP expression was observed in reconstituted bovine embryos by nuclear transfer with GFP-positive cells from 1-cell to blastocyst stages. These results show that it was possible to introduce the foreign genes into bovine embryos by nuclear transfer and these embryos show the expression of foreign gene in whole embryos.