No abstract

Abstract. Methylation of DNA in CpG islands plays an important role during fetal development and differentiation because CpG islands are preferentially located in upstream regions of mammalian genomic DNA, including the transcription start site of housekeeping genes and are also associated with tissue-specific genes. Somatic nuclear transfer (NT) technology has been used to generate live clones in numerous mammalian species, but only a low percentage of nuclear transferred animals develop to term. Abnormal epigenetic changes in the CpG islands of donor nuclei after nuclear transfer could contribute to a high rate of abortion during early gestation and increase perinatal death. These changes have yet to be explored. Thus, we investigated the genome-wide DNA methylation profiles of CpG islands in nuclei donor cells and NT animals. Using Restriction Landmark Genomic Scanning (RLGS), we showed, for the first time, the epigenetic profile formation of tissues from NT bovine fetuses produced from cumulus cells. From approximately 2600 unmethylated NotI sites visualized on the RLGS profile, at least 35 NotI sites showed different methylation statuses. Moreover, we proved that fetal and placental tissues from artificially inseminated and cloned cattle have tissuespecific differences in the genome-wide methylation profiles of the CpG islands. We also found that possible abnormalities occurred in the fetal brain and placental tissues of cloned animals.

Abstract. Abnormal development and fetal loss during postimplantation period are concerns for production of nuclear transferred animals. Aberrant DNA methylation is one of the reasons for poor survival of cloned animals. In mammalian genome DNA, CpG islands are preferentially located at the start of transcription of housekeeping genes and are associated with tissue-specific genes. The correct and consecutive mechanisms of DNA methylation in the CpG islands are necessary for selective gene expressions that determine the properties of individual cells, tissues, and organs. In this study, we investigated the methylation status of the CpG islands of the bovine Leptin and POU5F1 genes in fetal and placental tissues from fetuses produced by artificial insemination (AI) and nuclear transfer (NT) at days 48 and 59 of pregnancy. Altered DNA methylation was observed in the normal and cloned fetal, placental, and endometrial tissues using bisulfite sequencing and pyrosequencing. Different tissue-specific methylated regions in the bovine Leptin and POU5F1 genes show a variable methylation status in NT fetuses compared to AI control.

No abstract