| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2001: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Nuclear facotr kappa B (NF-kB) is a transcription factor and was originally described as being present in the cytosol of most most cell types as an inactive heterodimer composed of 50-kDa (p50) and 65-kDa (p65) subunits and bound to one of the IkB inhibitory proteins. After degrdation of IkB by its phosphorylation, NF-kB is activated and then p50 and p65 heterodimer translocates to the nucleus, where it directly binds its cognate DNA sequence. It has recently been found that NF-kB plays a pivotal role in morphogenesis and embryonic development, such as the formation of Drosophila ventral structures and chicken limb buds. However, the role of NF-kB in preimplantation development in mammals has not yet been clarified. In this study, the expression and the activation of NF-kB during preimplantation development in mice were first examined. Immunocytochemical studies using anti-p65 peptide monoclonal antibody showed intense cytoplasmic staining in embryos at the 1-cell to the blastocyst sta
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ges and that nuclear localization of p65, which is indicative of an active form of NF-kB was observed in 1-cell and 2-cell embryos. Gel shift analysis revealed that the DNA binding activity of NF-kB was found in embrys at the 1-cell and 2-cell stages, and the supershift analysis using anti-p50 and p65 antibodies confirmed that the active NF-kB in mouse 1-cell and 2-cell embryos is composed of p50 and p65 heterodimer. In the second experiments, effects of phosphorothioate antisense oligonucleotide (ODN) against p65 on in vitro development in mice were examined. Treatments with antisense ODN p65 of 1-cell embryos induced their developmental arrest at the 2-cell or 4-cell stages, whereas sense ODN p65 had no effects on their development. When embryos at the 2-cell stage were treated with antisense or sense ODN p65 , most embryos developed to the blastocyst stage. These results suggest that NF-kB plays an important role for regulation of development of preimplantation embryos. Furthermore, the identification of specific target genes under transcriptional regulation by NF-kB will definitely enhance our understanding of gene expression and development of preimplantation. Less
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