In-vitro manipulation of mammalian embryos

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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.