| Somatic cell nuclear transfer technology is an important milestone in the development of bioengineering technology which plays an important role in animal breeding, transgenic animals production, animal model establishing, artificial organs providing, rare and endangered animals protection and developmental biology. As a widely used experimental animal model, mouse gets the incomparable advantage than other organisms, so the use of mouse for somatic cell nuclear transfer technology research has very important theoretical and practical significance. But the efficiency of somatic nuclear transfer in mice is still low, each of the steps in the process of nuclear transfer has an important influence on the success or failure of the entire experiment, therefore optimize all links of the process of somatic cell nuclear transfer in mice has very important sense for improving the success rate of nuclear transfer. This paper mainly studies from three aspects:mouse oocytes parthenogenetic activation, somatic cell nuclear transplantation methods and tubal implantation of mouse embryos, aims to establish a mouse somatic cell nuclear transfer system, improve the efficiency of somatic cell nuclear transfer in mice and provide the technology foundation for other mammalian somatic cell nuclear transfer in our laboratory.Experiment1The Study of Mouse Oocytes Parthenogenetic Activation Methods1. In this study,18-hour oocyte of B6D2F1mouse was activated by electrical stimulation plus cytochalasin B (CB) treatment, to assess the effects of electric field strength, duration of electric pulse, and number of pulse on activation of the oocyte, as indicated by activation rate, cleavage rate and blastocyst formation rate after activation. The results showed that electrical stimulation at2.0kV/cm for80μs with3repeats could yield the highest activation rate (80.1%), cleavage rate (53.6%) and blastocyst formation rate (23.2%).2. On the basis of optimized electric stimulation parameters, we also tested the effect of10mmol/LSrCl2+CB treatment for4hours on activation of the18-hour oocytes. Treatment with Sr2+increased activation rate to82.5%, cleavage rate to56.1%and blastocyst formation rate to27.8%, and the conditions of the oocytes were better and developed into cleavage embryos and blastocysts of higher quality. So10mmol/LSrCl2+CB treatment for4h is ideal for parthenogenetic activation of oocytes.Experiment2The Study of the Somatic cell Nuclear Transfer Methods in Mouse1. Comparison of the oocyte enucleation methods:MII-phase oocytes were enucleated with the Piezo system using blind sucking, sucrose pretreatment for enucleation, and Hoechest33342staining method. The results showed that sucrose pretreatment for enucleation achieved the highest enucleation rate (82.7%), higher than that of blind sucking (49.0%) and Hoechest33342staining enucleation (80.4%). The sucrose pretreatment for encleation clearly shows the nuclear, so less cytoplasm is drawn and less damage is caused to the oocytes, and it is simple and easily operated. So sucrose pretreatment for enucleation is an ideal method for enucleation of oocytes.2. The effect of different concentrations of cytochalasin B (CB) on enucleation rate:oocytes with clearly visible nucleus was incubated in5μg/ml,10μg/ml, or20μg/ml of CB for enucleation, and enucleation rates were compared. The results showed that10μg/ml of CB had the highest enucleation rate (82.7%), significantly higher than that of5μg/ml and20μg/ml of CB (64.2%and51.3%respectively).3. Comparison of the ways to reconstruct an embryo:in this study, granulosa cells was used as donor of nucleus donor, and the nucleus was injected into cytoplasm or perivitelline space of the enucleated ovocytes, and embryo-reconstruction rate and embryo-cleavage rate of the two methods were compared. The results showed that cytoplasmic injection showed significantly higher rate of embryo reconstruction than perivitelline space injection (73.6%vs32.3%), and higher subsequent development potential, so cytoplasmic injection is better than perivitelline space injection. Experiment3The Establishment of Embryo Transfer Technology1. Effect of embryo age on tubal transplantation,1-cell,2-cell, and8-cell embryos of B6D2F1mouse were used for tubal transplantation, and pregnancy rate and calving rate of the surrogate mother were compared between the three to determine the optimum development stage for embryo transplantation. The results showed that transplantation using2-cell embryos has the highest pregnancy rate (60.0%) and calving rate (29.0%), significantly higher than that of1-cell embryos (pregnancy rate33.3%and calving rate13.6%) and8-cell embryos (pregnancy rate20.0%and calving rate7.3%).2. Comparison of two different sites of tubal transplantation:2-cell embryos were transplanted into fallopian tube through the wall or fimbriae of fallopian tube. The results showed that transplantation through incision on tubal wall had higher pregnancy rate and calving rate than tubal fimbriae transplantation (60.0%vs53.3%,29.0%vs26.3%). Moreover, the former way is simpler and cause less damage, to the fallopian tube and is thus an ideal way of embryo transplantation.In summary, fallopian tube wall-transplantation using2-cell embryo is the idea way of embryo transplantation. |
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