Effects Of Rabbit Anti-ES Cells Sera On Mouse Preimplantation Embryo

The technology of Tetraploid Embryos Complementation has been used in cloning mouse extensively in recent years. However, the tetraploid embryos have half cell numbers less that the diploid embryos, in the same stage, as well, the development potency is weaker. As a result, we help to make a kind of diploid embryos without ICM, replacing the tetraploid embryos, to improve this Complementation.Embryo stem cells (ES) were derived from the inner cell mass of preimplantation embryos by blocking the cells in their differentiation and culture program. A great deal of ES cells were collected and emulsified with adjuvants and injected into the body rabbit, collected the antisuerm, and incubated preimplantation mouse embryos in which. It would be inhibited the growth of ICM.The deduction is that the antiserum produced by rabbit may inhibit the development of the ICM, on the base of antigen-antibody response principle.We diluted the antiserum to 50, 100 and 200 times by KSOM medium, separately in the experiment. It demonstrated that the compaction of 8cell stage embryo was successfully inhibited, as well, the embryos showed fluid vacuoles in the blastula stage in 100 times diluted group; however there was no obvious effect in the 200 times diluted group. What’s more the nonimmune group did not appear differ from the blank medium group. We observed that the compaction was blocked by antiserum as the ratio of compaction was reduce to 70% when the embryos were cultured in the antiserum for 8 hours, and when the compacted embryos were explored in the antiserum, decompaction occurred, more over when the decompacted were prolonged beyond 32cell stage, the embryos result in an increasing proportion of malformed blastocysts.Incubation of the embryos in the antiserum for a long period resulted in the increasing incidence of two type blastocysts: (1) embryos showed fluid vacuoles,in which an unclear blastocoelic cavity was present, and which contained either no obvious ICM, but shows several fluid vacuoles: (2) True blastocysts, which appeared to have a normal morphology. When the two type were transplanted into the uterus of 2.5dpc pseudopregnancy female mouse, we put the mouse to death and collected the implantation condition after 10days. The result demonstrated that, both of the fluid vacuoles group and normal morphology group showed a extreme Significant difference(p<0.01), compared with the control group respectively, however there was no significant difference between Interclas(sp>0.05). But, even in the successfully implanted embryos in the antiserum group, most of the fetuses had become vestigial, only with, non-absorbed tissue mass look like the Early placenta, left.We Immunohistochemistry stained the embryos using Hoechst33342 after the 8cell incubated in the antiserum for 60 hours. Counting the cell numbers showed that there was a significant decrease in antiserum group(p<0.01).Embryonic inoculation test appeared that, the number of Outgrowth came from antiserum incubation was significant lower than the control group, well, the morphology performed more separated, the stereoscopic was in default strive, the area is lesser and the edge is not tidy enough. Especially, it appeared some exceptions which to be only with TE cells but ICM in the feeders in antiserum treated group.Immunohistochemistry stained the Outgrowth through alkaline phosphatase and embryo pluripotency factor Oct4 did appear that the pluripotency of the embryos which continuously exposed to antiserum were still exist, this result was taken as an indication of the existence of ICM, however it subjected a decrease in cell numbers and a obviously disordered distribution.