A Preliminary Investigation On Mouse Tetraploid Embryo Complementation

Embryonic stem (ES) cells are drived from the inner cell mass (ICM) of balstocysts with potential capacity of self-renewal and differentiation. ES cells possess the capacity of differentiation to various somatic cell lineages, so they are called pluripotent stem cells. The diploid embryo of mice may produce tetraploid embryo by electrofusion. The tetraploid embryo whose developmental capacity is the same as parthenogenetic and androgenesis, can't develop into normal individuals. During the formation of chimeras of ESCs and tetraploid embryos, ESCs participate widely the formation of embryo allantoic, Amniotic chorionic rnesoderm and yolk sac mesoderm. While the origin cells of tetroploid distributed mainly in the extraembryonic tissues like rophoblast, yolk sac membrane, chorioallantoic membrane, placenta, hardly participate the formation of fetus itself. If the tabling of ESCs and tetraploid embryos compensate their developmental capacity mutually, We would get the individual totally developed from ESCs. This technology is named tetraploid embryos complementation and the mice obtained are called ES mice. Nagy obtained ES mice which completely developed from ESCs by tetraploid embryos aggregation in 1993.Then more scientists got ES mice by tetraploid blastocyst injection.1.Comparison of developmental capacity of tetraploid embryos produced by electrofusion. We have used Kunming mice embryos in 2-cell stage to obtained the tetraploid blastocysts by electrofusion, and explored the electrical fusion conditions. The result showed that under the conditions of AC 1-2 voltage sorting, DC 50 voltage electric shock, pulse duration 35μs, pulse 2 times,2-cell fusion rate will reach to 99.5%, blastocysts rate will reach to 74.7%. There wasn't significant difference compared with normal 2N blastolyst rate(80.2%). It showed that tetraploid embryos' developmental capacity is equal to the normal blastolysts.2. In vitro culture of Green fluorescent stem cells and identification.We can use domestic mistomycin to deal with mice fibroblast cell as feeder layer to inoculate embryonic stem cells. The condition is that the concentration of mitomycin must be 30 mg/ml and processing must be 4h. Cultured ESCs by the conventional method, ESCs perfect medium must be DMEM containing 0.1mmol/Lβ-mercaptoethanol,50v/mL penicillin,50mg/L streptomycin,15% fetal bovine serum, 1000v/mL LIF. ESCs grow on feeder layer with cloned-like shape. Stained with alkaline phosphatase, SSEA-1 and OCT-4, the ESCs are identified as positive. It shows that ESCs can be used for subsequent experiments.3.Blastocyst injection and embryo transfer of ESCWe can inject the stem cells into blastocysts by Eppendorf micromani-pulation system, with 15 ESCs every tetraploid. After injection, we can transplant the embryos into 2.5day-pseudopregnant uterus of female mice, with 10 embryos every side, then we can wait for the natural delivery or caesarean section. At last, after injecting ESCs into tetraploid blastocysts, we can see Green fluorescent ESCs incorporate into the inner cell mass. After transplanting ESCs into 2.5day-pseudopregnant uterus of female mice, the uterus will be enlarged and Green fluorescence can be seen in the endometrial but mice have not be born. It shows that the embryos are absorbed at some stage after implantation.