| Chimera is an individual with a double chromosome complement which is aggregated or injected by embyos or cells with different hereditay. Chimera technique is very important in developmental biology research, chimeric offspring has become the accreditation standards that validated the developmental potential of pluripotent stem cells (embryonic stem cells, induced pluripotent stem cells). Study of chimeric mice is extensive and in-depth in mammals, but few studies about other species. Pig is an important species in livestock production, human medicine model, porcine pluripotent stem cell research, and getting more and more attention in recent years. As an important criterion for stem cell pluripotency testing, porcine chimerism research also got everyone’s attention. Although some reports about porcine chimera have been published, relatively there is no systematic study and not enough great importance to the special laws of the porcine embryonic development. So the experiments first established the technical system of lentivirus mediated green fluorescent protein(GFP) transgenic embryos. Furthermore, to explore chimeric development law between different developmental embryos stages and the ability of the iPS cell chimeric development, for providng a solid foundation for the future research.The results were as follows:1. Study on lentivirus mediated green fluorescent protein(GFP) transgenic embryos(1) In groups of2×109I.U./ml lentivirous injecteion in the perivitelline of the same stage of the embryos, the infection rate and GFP-positive blastocyst rate of IVF(80.00%,90.74%) and parthenogenetic embryos (76.36%,89.56%) were significantly higher than other groups (P<0.05), The fluorescence intensity of the blastocyst stage was also significantly higher than other groups.2×108I.U./ml lentivirous injecteion the embryo efficiency followed by, efficiency of2×lO8I.U./ml lentivirous is the lowest.(2) The same titer lentiviral infection different periods embryonic, in groups of2×109I.U./ml lentivirous injecteion in the perivitelline of the2-cell stage embryos, the infection rate and GFP-positive blastocyst rate of IVF(80.00%,90.74%) and parthenogenetic embryos (76.36%,89.56%) were significantly higher than the infection rate and GFP-positive blastocyst rate of IVF(60.07.00%,57.51%) and parthenogenetic embryos (79.17%,75.29%, P<0.05), The fluorescence intensity of the blastocyst stage was also significantly higher than the1-cell stage embryos. We get the same results in the group of2×108I.U./ml lentivirus injected the both stage embryos. (3) When the lentiviral titer is less than2×107I.U./ml, even injection at the2-cell embryos is almost impossible to effectively infect porcine embryo, and there is no significant difference in cleavage rate, blastocysts rate, blastocysts cell number compared with the control group.2. Different stages of embryo aggregation(1) The chimeric rate of embryos derived from aggregating of two8-cell embryos (75.00%) were significantly higher than that of these embryos derived from aggregating of two4-cell embryos (65.00%) or2-cell embryos (53.80%).(2) The chimeric rate of embryos derived from aggregating of two2-cell stage embryos (53.85%) and embryos from blastomere exchanging of2-cell stage embryos (62.50%) were significantly higher than that of aggregating of2-cell with4-cell or8-cell embryos (18.60%, P<0.05).3. iPS chimeric embryos(1)Injection iPS with gfp transgene to2-cell4-cell and8-cell embryos, cultured for1-2days, we did not observe green fluorescent, chimeric embryo can not be formed. Injection in the blastocyst stage, after1-2days in vitro culture, some of the ips cells can contribute to the blastocyst, to form chimera.(2) Comparing the ips chimeric rate, No.68, No.120and122iPS cell are no significant differences. In the experimental group, the average cell number of blastocysts were higher than the control group, indicating that some of the ips cells already aggregation with embryos.(3) Analysis of the results after embryo transfer, four iPS cells as donor cells, in vivo and IVF embryos as a recipient, transplanted a total of19sows of which No.68chimeric embryo transplant recipients occurred pregnancy, but probably40days abortion, the carcass was absorbed getting two placentas.(4) The identification and analysis of the placenta, the porcine placenta dispersed and was observed the villi and villous capillary by the tissue sections with HE staining. iPS contribute to the placental tissue by PCR analysis.In summary,2×109I.U./ml of lentivirous injection in the perivitelline of two-cell stage embryos shows the highest infection efficiency and has no significant effect on developmental of porcine embryos. The chimeric rate of embryos derived from aggregating two8-cell embryos is higher than the others.The chimerism rate of the synchronized developmental embryos is higher than that of the non-synchronized embryos. The stage of blastomeres injected with iPS can not formed chimeric embryos, but blastocyst injection. Preliminary analysis shows that the iPS cells contribute to the placenta. |
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