Study On The Technology And Mechanism Producing Mice Derived From Embryonic Germ Cells Aggregated With Tetraploid Embryos

The completion of the Human Genome Project marks the beginning of a new era in functional genome. Gene targeting technology plays important roles in elucidating physiological function of genes in vivo and applications in the model animals. The gene targeted mice are commonly produced by breeding the heterozygous targeted mice following microinjecting the homologous recombinant ES cells into the blastocoele cavity of 3.5dpc embryos at the blastocyst stage to give birth to chimeric mice. But the heterozygous targeted mice can directly be generated by the technology of tetraploid embryo complementation, which is a simple and efficient production of heterozygous targeted mice derived from the homologous recombinant ES cells aggregated with tetraploid embryos. So it will save much time to produce the targeted mice with this method for studying the gene function and make the medical animal model.To establish the platform producing the mice derived from embryonic germ cells aggregated with tetraploid embryos, the effects of the aggregation methods, the culture vector and the number of EG cells on producing the chimeric embryos were studied and the best methods were determined according to the standards of ICM/Total cell numbers and the index of cell apoptosis.Then the experiment discussed the effects of the fetal age and the feeder cells on isolation and culture of mice EG cells and got the results that MEF feeder and 11.5～12.5dpc PGCs were more suitable to isolate and culture EG cells. At the same time, the study got the results that there were no differences of the DNA methylation levels in the differential methylation region of the imprinted genes H19 and Igf2 in the different fetal age EG cells, the different individual EG cells and the different passaged EG cells.In addition to, the quality of the tetraploid embryos was worse than the diploid embryos in vivo and in vitro according to test the ICM/Total numbers and the index of cell apoptosis. In another study, the DNA methylation patterns of the mouse tetraploid embryos were investigated by immunofluorescence staining with an antibody against 5-methylation and the aberrant methylation levels of the tetraploid embryos may lead to subsequence developmental failure and embryo death. Finally, the expression and the methylation of the imprinted Igf2 and H19 genes in mouse tetraploid blastocysts were studied and the findings indicated that genome duplication may be involved in the aberrant expression and methylation levels of imprinted Igf2 and H19 genes in tetraploid blastocysts in vitro, and that this may cause the later developmental failure of tetraploid embryos.