Role of transcription factor, NF-kB in preimplantation development and zygotic gene activation in mice

转录因子 NF-kB 在小鼠植入前发育和合子基因激活中的作用

• 批准号: 13660287
• 负责人: YAMADA Masayasu
• 金额: $ 2.18万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660287/
• 关键词: NF-kB; Mouse embryo; Nuclear localization; DNA binding; Gel shift assay; Antisense oligmer; Preimplantation development; P65-p50 heyerodimer; 免疫蛍光染色; 核内への局在; RelA-p50二量体

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 … More 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

核转录因子kB是一种转录因子，最初被描述为存在于大多数类型细胞的胞浆中，是一种由50 kDa(P50)和65 kDa(P65)亚基组成的非活性异源二聚体，与IKB抑制蛋白之一结合。当IKB被其磷酸化降解后，核因子-kB被激活，然后p50和p65异源二聚体移位到细胞核，在那里它直接与其同源DNA序列结合。最近发现，核因子-kB在形态发生和胚胎发育中起着关键作用，如果蝇腹侧结构的形成和鸡肢芽的形成。然而，核因子-kB在哺乳动物着床前发育中的作用尚未阐明。本研究首先检测了小鼠着床前发育过程中核因子-kB的表达和激活情况。用抗p65多肽单抗进行的免疫细胞化学研究显示，在1-细胞到囊胚期…的胚胎中胞浆染色很强。在1-细胞和2-细胞胚胎中观察到更多的GES和p65的核定位，这表明核转录因子-kB是一种活跃的形式。凝胶位移分析表明，核因子-kB的DNA结合活性存在于1-细胞期和2-细胞期，抗p50和p65抗体的超移位分析证实，小鼠1-细胞和2-细胞胚胎中具有活性的核因子-kB由p50和p65异源二聚体组成。在第二个实验中，检测了针对p65的硫代反义寡核苷酸(ODN)对小鼠体外发育的影响。反义ODN p65处理的1-细胞胚胎发育停滞于2-细胞期或4-细胞期，而正义ODN-P65对其发育无影响。用反义或正义ODN p65处理2-细胞期胚胎时，大多数胚胎发育到囊胚期。这些结果表明，核因子-kB在着床前胚胎发育过程中起着重要的调节作用。此外，识别核因子-kB转录调控下的特定靶基因，将有助于我们更好地了解植入前基因的表达和发育。较少

项目成果

Kurosaka S.: "Dependence of DNA synthesis and in vitro development of bovine nuclear transfer embryos on the stage of the cell cycle of donor cells and recipient cytoplasts"Biol. Reprod.. 67. 643-647 (2002)

Kurosaka S.：“牛核移植胚胎的 DNA 合成和体外发育对供体细胞和受体细胞质的细胞周期阶段的依赖性”Biol。

Nishikimi A.: "Localization of nitric oxide synthase activity in unfertilized oocytes and fertilized embryos during preimplantation development in mice"Reproduction. 122. 957-963 (2001)

Nishikimi A.：“小鼠植入前发育过程中未受精卵母细胞和受精胚胎中一氧化氮合酶活性的定位”复制。

Matsukawa T.: "Alleviation of the two-cell block of ICR mouse embryos by polyaminocarboxylate metal chelators"Reproduction. (印刷中).

Matsukawa T.：“通过聚氨基羧酸盐金属螯合剂缓解 ICR 小鼠胚胎的两细胞阻滞”（正在出版）。

Nishikimi A., et al.: "Nuclear translocation of a pre-mRNA splicing factor, p100^<*****>, in mouse 1-cell embryos"J. Reprod Dev.. 48. 257-263 (2002)

Nishikimi A. 等人：“小鼠 1 细胞胚胎中前 mRNA 剪接因子 p100^<*****> 的核易位”J.

Matsukawa T.: "Alleviation of the two-cell block of ICR mouse embryos by polyaminocarboxylate metal chelatars."Reproduction. 124. 65-71 (2002)

Matsukawa T.：“聚氨基羧酸金属螯合剂减轻 ICR 小鼠胚胎的两细胞阻滞。”复制。