| Somatic cell nuclear transfer (SCNT), as a hot spot of the life science, has achieved dramatic advancement in many mammals. However, the whole efficiency of the traditional micromanipulation was very low. Moreover, the procedure was very complex and required expensive equipments and considerable technical skills. All this restricted the widespread application of this technology. Recently, there had been developed a new nuclear transfer method---hand-made cloning(HMC) technology derived from the chemical induced enucleation technology and the zona-free nuclear transfer technology, which simplified from the enucleation of oocytes and the fusion of the somatic cell and oocytes, respectively. The HMC technology not only avoided trivial procedure and reduced costs, but also made the fusion of the oocyts and somatic cell easy. So this study did some experiments combining the chemical induced enucleation technology and the zona-free nuclear transfer technology using the ICR mouse oocytes as materials.1. Oocytes enucleation was one of the key factors to decide the successes of animal cloning.This study optimized the protocol of demecolcine(DC)-induced enucleation using the ICR mouse oocytes as materials in the basic of the gained achievement of abroad and our laboratory, which examined the effects of oocytes age, activating methods and DC initial treatment on the demecolcine-induced enucleation rate. The result showed that: Whatever activated by 7% ethanol or 10mM SrCl2, the induced enucleation rate of oocytes collected at 17～18 h after hCG administration was significantly higher than collected at 14～15h and 23～24h after hCG administration(P<0.05), but lower slightly than collected at 20～21h after hCG administration(P>0.05); The blastocyst development rate of activated oocytes collected at 17～18 h after hCG administration was significantly higher than other groups(P<0.05). The second PB2 extrusion rate of oocytes activated by 10mM SrCl2 had no significant difference at different time after hCG administration (P>0.05), but the second PB2 extrusion rate of ethanol groups was raising first , then lowing as the raising of the time after hCG administration. There was no significant difference between ethanol groups and SrCl2 groups on the induced enucleation rate, the second PB2 extrusion rate and the blastocyst development rate of oocytes collected at the same time(P>0.05). The induced enucleation rate of ethanol groups treated with demecolcine at immediately post-activation was significantly higher than groups treated with demecolcine at 10 and 15 minutes post-activation(P<0.05), but no difference with the group treated with demecolcine at 5 minutes post-activation (P>0.05). The induced enucleation rate of SrCl2 groups treated with demecolcine after 15 minutes post-activation was significantly higher than groups treated with demecolcine after 25 and 35 minutes post-activation(P<0.05), but no difference with the group treated with demecolcine at 0 minutes post-activation (P>0.05). The results indicated that oocytes collected at 17～18 h after hCG administration were favoriate to demecolcine-induced enucleation; The whole efficiency of 10mM SrCl2 used for demecolcine-induced enucleation on ICR mouse was better slightly than 7% ethanol; The highest induced enucleation rate was acquired while ethanol-activated oocytes treated with demecolcine at immediately post-activation(63.4%), However, the highest induced enucleation rate was acquired while SrCl2-activated oocytes treated with demecolcine after 15 minutes post-activation(63.9%).2. The culture of zona-free embryos was the issue that must have been solved before carrying the zona-free nuclear transfer technology. To search for an effective culture method for zona-free embryos and provide technical support for handmade cloning, this study contrasted three culture methods for the zona-free embryos of mouse, then modified the well of the well (WOW) culture method by embedding the well with calcium alginate gel and tried to combine with the co-culture technology. The results showed that the blastocyst development rate(54.7 %) and the average cell numbers of blastocysts (54.3) culturing with the calcium alginate gel embedding well(AEW) culture method were significantly higher than those of culturing with the micro-drops single(MDS) culture method and agarose-pillar embedding(AE) culture method (P<0.05), but no significant differences (P>0.05) with the WOW culture method(P>0.05); the optimal concentrations of the sodium alginate and calcium ion were 0.7 % and 1.5 % respectively; the cleavage rate(89.7 %) and the blastocyst development rate (67.9 %) culturing with the AEW culture method combining with co-culture technology were significantly higher than those of culturing with the AEW culture method alone (P<0.05). The results indicate that the efficiency of AEW culture method is better than that of the other culture methods, combining with granule cells co-culture technology can greatly improve the cleavage rate and the blastocyst development rate in culturing zona-free embryos.3. To simplify the procedure of the somatic cell nuclear transfer technology and raise the efficiency of the nuclear transfer, this study set up a set of zona-free somatic cell nuclear transfer system to suit ICR mouse combining the chemical induced enucleation technology and the zona-free nuclear transfer technology using the ICR mouse oocytes as materials. The results showed that: the fusion effect using single direct current pulse of 95V/mm electrical field was significantly higher than that of 80V/mm group and 110V/mm group(P<0.05); The activation rate(82.9%) of treating with 5mM SrCl2 for 1h group was significantly higher than other groups(P<0.05), and the regrade rate(10.3%) of treating with 5mM SrCl2 for 1h group was significantly lower than other groups(P<0.05). However, the blastocyst development rate(46.4%) of treating with 5mM SrCl2 for 1h group was slightly higher than other groups(P>0.05); The times spending in the zona-free nuclear transfer was significantly shorter than that spending in the traditional nuclear transfer(P<0.05); The reconstruction rate of reconstructed embryos(83.3%) using zona-free nuclear transfer was no difference with that using traditional nuclear transfer(P>0.05); The development of reconstructed embryos using zona-free nuclear transfer was lower than that using traditional nuclear transfer, but no significant difference(P>0.05). The results indicated that: the best fusion effect was acquired when using single direct current pulse of 95V/mm electrical field; the best activation effect was acquired when oocytes were treated with 5mM SrCl2 for 1h; The global efficiency of zona-free nuclear transfer system this study set up was higher than traditional nuclear transfer.However, the steps of this system need to be consummated to raise the blastocyst development rate.
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