In-vitro manipulation of mammalian embryos

在本项目中，我们对哺乳动物胚胎的冷冻保存和克隆进行了大量的研究，包括小鼠和大型家畜(羊和牛)。我们建立了一种新的快速冷冻程序，使用渗透性冷冻保护剂3M乙二醇和0.25M非渗透性糖(乳糖、蔗糖或海藻糖)的混合物。这一简单的冷冻程序不仅对小鼠胚胎和卵母细胞的冷冻非常有效，而且对绵羊胚胎也是如此。我们还研制了两种新型玻璃化液，分别由25%乙二醇和25%DMSO和20%乙二醇、20%DMSO和10%1，3-丁二醇组成。这两种玻璃化液不仅使小鼠和牛胚胎具有较高的体外存活率，而且在将玻璃化冷冻胚胎移植给受体动物后，产生了大量的后代，包括小鼠和小牛。在胚胎的克隆方面，我们利用小鼠卵母细胞和胚胎进行了大量的实验，以确定影响核移植胚胎重编程的因素。我们的结果中最显著的发现之一是，当从供体胚胎的核移植到去核的卵母细胞时，供体细胞周期阶段对核移植胚胎的染色质结构和发育有着至关重要的影响。通过移植早期细胞核，来自2、4或8细胞期胚胎的核移植胚胎获得了高比例的囊胚发育和后代。这是第一次取得这样的成果。

In this project, we have performed a lot of studies on cryopreservation and cloning of mammalian embryos including mouse and large domestic animals such as sheep and cattle.We established a new rapid freezing procedure using a mixture of permeating cryoprotectant such as 3M ethylene glycol and 0.25 M non-permeating sugar (lactose, sucrose or trehalose). This simple freezing procedure was demonstrated to be very effective not only for cryopreservation of mouse embryos and oocytes, but also for sheep embryos. We also developed two types of new vitrification solutions consisted of 25% ethylene glycol and 25% DMSO; and 20% ethylene glycol, 20% DMSO and 10% 1,3-butanediol, respectively. These two vitrification solutions gave not only a high in-vitro survival for mouse and cattle embryos, but also resulted in many offspring, both mice and calves, after the transfer of vitrified-warmed embryos to recipient animals.Regarding the cloning of embryos, we have done a lot of experiments using mouse oocytes and embryos to determine the factors affecting reprograming of nuclear tranplanted embryos. One of the most salient findings from our results is that the donor cell-cycle stage critically affects the chromatin structure and development of nuclear transplant embryos when the nuclei from donor embryos were transferred to enucleated oocytes. With transplantation of early-stage nuclei, high proportions of development to the blastocyst and of offspring were obtained from nuclear transplant embryos with a nucleus from a 2-, 4- or 8-cell-stage embryos. This is the first time that such result has been achieved.