The Study On Somatic Nuclear Transfer Of Mouse Assisting With Demecolcine Induced Enucleation

Karyoplast and cytoplast are two important components of reconstructed embryos in the nuclear transfer. Especially,the latter is much critical to reprogramming of nuclear and development of embryos. At present, cytoplasts are obtained by removing the chromatin using micromanipulation, but this method has some shortcomings, such as time-consuming, technical demanding and damaging to cytoplasts etc. Recently, some scientists try to induce the extrusion of chromatin using reagent, which was named simply induced-enucleation (IE). Several overseas labs have made series of studies on IE, but they didn't analyze all kinds of effecting factors systematically on the IE rate. Meanwhile, the domestic report related to IE hasn't been observed. So establishing optimized IE procedure is the main aim of this research. Through the fundemental study on Kunming mouse, we can obtain some experiences and datas, which are useful to oocytes enucleation of large animals. On the basis of exiting technique pathway of demecolcine-induced enucleation(IE),several factors(Demecolcine concentration,time of demecolcine inition and treatment,oocytes age) affecting the IE rate were tested using Kunming mouse oocytes. The experiments' results demonstrated that: The IE rate of activated oocytes treated with KSOM medium containing 0.4μg/mL or 0.5μg/mL demecolcine for 60 min is 27.3% and 33.3% respectively, and difference is not significant between two groups, but compared with 0.6μg/mL group, the difference is significant. The higher IE rate(24.5%~31.9%) were obtained when oocytes were exposed to 0.5μg/mL demecolcine between 0 and 5 min postactivation and treated for 60-180 min. The maximum IE rate was 31.9% when oocytes were treated with demecolcine postactivation 0 min for 60 min, which differed significantly with 30 min group,compared with 90 min and 180 min group, the difference is not significant.The relative lower IE rate(5.7%~12.0%) were obtained when oocytes were treated with demecolcine postactivation 10-15 min for different time. The IE rate of oocytes collected from Kunming mouse at 17~18 h after hCG administration is 27.1%, which is significantly higher than 14~15 h and 20~21 h group (7.3%,14.1%). The fundamental study on cumulus nuclear transfer of mouse was made on the basis of optimized IE procedure. The development rate of different phase is 12% and 2% respectively. This experiment firstly assayed the enucleation of oocytes of Kunming mouse induced by demecolcine and somatic nuclear transfer of mouse which is assisted with optimized IE procedure.