Study of implantation using molecular biological means using xeno-chimeric embryos.

我们利用分子生物学技术和转基因动物研究了植入的机制。我们已经建立了绿鼠和红鼠两条线。这些Zeno-clilmeric胚胎的组合使我们能够区分细胞来自哪个物种的贡献。为了从大鼠胚胎中获得足够的来源，我们建立了由“绿鼠”骨髓细胞(MAPC)培养的多能干细胞。我们还成功地建立了滋养外胚层来源的干细胞。在生产嵌合小鼠的过程中，我们利用X染色体上标记了绿色荧光蛋白(X^*)的胚胎产生了XX7XY嵌合体，使得荧光绿色雌性(XX^*)生殖细胞很容易与非荧光雄性生殖细胞(XY)区分开来。利用绿色荧光蛋白标记技术，从精巢中分离出XX个“成熟原细胞”，并对其基因组印迹状态进行了研究。结果表明，这些XX细胞经历了父系印记，尽管它们的染色体组成。正如之前在性反转XXsxr睾丸中所指出的，我们还发现在XX^*7XY嵌合睾丸的生精小管内有少量绿色的XX^*生殖细胞发育成“卵”。这些细胞在出生时与XX^*成熟原细胞难以区分，但在睾丸环境中恢复了卵子发生。通过从嵌合测试中回收卵子来检验“睾丸卵子”的生物学性质。睾丸卵子不仅形成了卵子特有的结构--透明带，而且能够与精子融合。收集的睾丸卵子被表明接受了母体印记，尽管睾丸环境如此。基因组印记并不总是遵循生殖细胞所在的环境条件；相反，它是由生殖细胞在早期胚胎阶段选择的性别决定的。

We investigated the mechanism of implantation using molecular biological techniques and transgenic animals. We have established "green rat" lines together with "red mouse" lines. The combination of these zeno-clilmeric embryos allows us to differentiate the contribution of cells from which species the cells are derived from.In order to have a sufficient source from rat embryos, we have established multipotent stem cells from "green rat" bone marrow cells (MAPC). We also succeeded to establish the stem cells of trophectoderm origin.In the middle of producing chimeric mice, We produced XX7XY chimeras by using embryos whose X chromosomes were tagged with EGFP (X^*), making the fluorescent green female (XX^*) germ cells easily distinguishable from their nonfluorescent male (XY) counterparts. Taking advantage of tagging with EGFP, the XX^* "prospermatogonia" were isolated from the testes, and the status of their genomic imprinting was examined. It was shown that these XX cells underwent a paternal imprinting, despite their chromosomal constitution. As previously indicated in sex-reversal XXsxr testes, we also found a few green XX^* germ cells developed as "eggs" within the seminiferous tubules of XX^*7XY chimeric testes. These cells were indistinguishable from XX^* prospermatogonia at birth but resumed oogenesis in a testicular environment. The biological nature of the "testicular eggs" was examined by recovering the eggs from chimeric testes.The testicular eggs not only formed an egg-specific structure, the zona pellucida, but also were able to fuse with sperm. The collected testicular eggs were indicated to undergo maternal imprinting, despite the testicular environment. The genomic imprinting did not always follow the environmental conditions of where the germ cells resided ; rather, it was defined by the sex that was chosen by the germ cells at early embryonic stage.