Methylation Of H19 Gene In ES Mice And Embryonic Stem Cells

Mouse embryonic stem (ES) cells and tetraploid embryos have different distribution. The yolk sac endoderm and the trophoblast cell lineages are tetraploid embryo-derived, whereas the embryo proper, the amnion, the yolk sac mesoderm, the allantois, and the chorionic mesodermderived part of the placenta are completely ES cell-derived. Embedding ES cells in tetraploid embryo, we can obtain the ES mouse completely from ES cells with the method of tetraploid embryo complementation.Because of the abnormal epigenetic state of donor genome, all of cloned animals display a range of developmental disfigurements. And the clones which survive to adulthood or live longer only have less abnormalities than others. Abnormal phenotypes of cloned offspring correlate with faulty epigenetic reprogramming during reconstructed embryo early development. Faulty epigenetic reprogramming in cloned embryo leads to irregularities in gene expression that might result in the abnormal phenotypes in cloning. DNA methylation which is one of important epigenetic modifications in eukaryote genome is a main instrument of regulating genome function and plays a prominent part in embryonic normal development. Through researching the pattern of DNA methylation, we found that there were many abnormal DNA methylation patterns in cloning, which might be the primary reasons for inducing early death of cloned embryos and developmental abnormalities in cloning. And we knew that the irregularities in imprinted gene expression which regulated by DNA methylation could lead to developmental abnormalities in cloning. So we deduce that the methylation state of imprinted gene in newborn dead clonings might appear something changes comparing with controls.Three days after ovulation, 37 blastocysts were recovered by a surgical uterine flush technique from C57BL/6J×129/Sv mice and played on passage 2 to passage 5 mouse(ICR) embryonic fibroblasts feeder layer mitotically inactivated with mitomycin C(1mg/100mL for 4h). After 4 days, the intact inner cell mass (ICM) was separated with 0.25% trypsin-EDTA and again seeded on feeder in Dulbecco's modified Eagle medium (4500mg of glucose per liter) supplemented with 15% fetal bovine serum, 0.1mM nonessential amino acids, 0.1mM 2-mercaptoethanol, 2mM glutamine, 50units/mL of penicillin, and 50units/mL of streptomycin, 1000IU/mL Leukemia Inhibitory Factor (LIF). Cultures were grown in 5% CO2, 95% humidity and 37℃in incubator. After 6 to 7 days of culture, three ES cell lines were selected and explanted.Genomic DNA was extracted from the tails of normal adult mice, normal newborn mice, adult ES mice and newborn dead ES mice with buffer saturated phenol. The methylation status of two loci at 5'UTR region in imprinted gene H19 of them were detected in turns using methylation-sensitive restriction endonuclease-PCR technique. The results presumed that the methylation status of imprinted gene H19 in adult ES mice are identical to that of normal adult or newborn mice. However, some significant differences in the methylation status of imprinted gene H19 were found between newborn dead ES mice and the others.By the same principle, we purified ES cells (C57BL/6J×129/Sv) in nicer state to research. From the outcomes, we guessed that the methylation status of imprinted gene H19 in ES cells were probably different from that of adult ES mice, normal adult mice and normal newborn mice, and there seemed no notable difference among different passage ES cells.