Epigenetic analysis of bovine parthenogenetic embryonic fibroblasts

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  • KANEDA Masahiro
    Division of Animal Life Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
  • TAKAHASHI Masashi
    Department of Animal Science, Graduate School of Agriculture, Hokkaido University, Hokkaido 060-8589, Japan
  • YAMANAKA Ken-ichi
    Faculty of Agriculture, Saga University, Saga 840-8502, Japan
  • SAITO Koji
    Kumamoto Prefectural Agriculture Research Center, Kumamoto 861-1113, Japan
  • TANIGUCHI Masanori
    Kumamoto Prefectural Agriculture Research Center, Kumamoto 861-1113, Japan
  • AKAGI Satoshi
    Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan
  • WATANABE Shinya
    Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan
  • NAGAI Takashi
    Headquarters, National Agriculture and Food Research Organization, Ibaraki 305-8517, Japan

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<p> Although more than 100 imprinted genes have already been identified in the mouse and human genomes, little is known about genomic imprinting in cattle. For a better understanding of these genes in cattle, parthenogenetically activated bovine blastocysts were transferred to recipient cows to obtain parthenotes, and fibroblasts derived from a Day 40 (Day 0 being the day of parthenogenetic activation) parthenogenetic embryo (BpEFs) were successfully obtained. Bovine embryonic fibroblasts (BEFs) were also isolated from a normal fertilized embryo obtained from an artificially inseminated cow. The expression of imprinted genes was analyzed by RT-PCR. Paternally expressed genes (PEGs) in mouse (viz., IGF2, PEG3, ZAC1, NDN, DLK1, SGCE, and PEG10) were expressed in BEFs, but not in BpEFs, suggesting that these genes are also imprinted in cattle. However, other PEGs in mouse (viz., IMPACT, MAGEL2, SNRPN, and PEG1/MEST) were expressed in both BEFs and BpEFs. These genes may not be imprinted in BEFs. The expression of seven maternally expressed genes in mouse was also analyzed, and only CDKN1C was not expressed in BpEFs. The DNA methylation patterns of repetitive elements (Satellite I, Satellite II, alpha-satellite, and Art2) were not different between the BEFs and BpEFs; however, the differentially methylated region (DMR) of paternally methylated H19 was hypomethylated, whereas those of maternally methylated PEG3 and PEG10 were hypermethylated in BpEFs, as expected. The methylation of the SNRPN DMR was not different between the BEFs and BpEFs, in accordance with the SNRPN expression levels in both cell types. The XIST gene, which is essential for X chromosome inactivation in females, was expressed in BpEFs, whereas its DMR was half-methylated, suggesting that X chromosome inactivation is normal in these cells. Microarray analysis was also applied to identify novel PEGs that should be expressed only in BEFs but not in BpEFs. More than 300 PEG candidate genes, including IGF2, PEG3, and PEG10, were obtained. These results illustrate the epigenetic characteristic of bovine parthenogenetic embryos and contribute to the identification of novel imprinted genes in cattle.</p>

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