Studies On Nuclear Transfer Using Somatic Cells As Nucleus Donors In Mice

1. Seven different media, in which glucose, phosphate, EDTA and taurine were added or excluded, were applied to the culture of early embryos of ICR mice in vitro. The results showed that glucose and phosphate did harm to the development of early embryos. When the glucose and phosphate were excluded, the rates of embryos overcoming the 2-cell block were improved. Once 2-cell block was overcome, glucose started to enhance development of early embryos. The medium, in which taurine was added, was showed to be very effective to promote overcoming 2-cell block. EDTA was not advantageous by itself, but can coordinate taurine to promote development of mice early embryos. The modified media mM16+T+E and mCZB+T both benefited the mice early embryos' development, and could be used as medium to culture reconstituted nuclear transfer embryos.2. For the parthenogenetic activation hi mice oocytes, the effects of concentration and duration of alcohol were studied. The results showed that the treatment of 7% alcohol for 5 minutes was the best. The oocytes age did not significantly affect the parthenogenetic activation rates between 18h~23h, but the rates became lower as the oocytes aging. For the reason of postponed activation in reconstituted embryos of nuclear transfer, 21h was chosen as age of parthnogenetic activation. On electro activation, there was no significant difference between the voltage of 80v/mm, 100v/mm and 120v/mm; the activation rate of 80v/mm was the highest. The activation rate rose with the increase of duration pulse, 80 u s got the best rate. No difference was showed between treatment of Sr2+, 7% alcohol for 5minutes and electro activation of lOOv/mm for 80 s, they all got high activation rate. The treatment of Sr2+ is simply and high rate, and so it was chosen as the way of parthenogenetic activation on reconstituted embryos of nuclear transfer.3. Three procedures of nuclear transfer in mice were studied. They all referred the first polar body to orient for enucleating the nuclei, so the rates of nuclei enucleation were not different. Oocytes were extraordinarily sensitive to the conventional micromanipulation, which generally resulted in cell lysis. Cutting zona pellucida beforehand could makemanipulation more softly to raise the live rate of manipulated oocytes. Pizeo-acuated micromanipulation is the best method for mice nuclear transfer, but it needs more delicate manipulation, which is not easy to master. Intracytoplasmic nuclei injection is more effective than cell electro-fusion method on reconstituting embryos of nuclear transfer.