Study on ECM tenascin-family proteins by gene knockout

ECM腱蛋白家族蛋白基因敲除研究

• 批准号: 07044209
• 负责人: IKEMURA Toshimichi
• 金额: $ 1.34万
• 依托单位: NATIONAL INSTITUTE OF GENETICS
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044209/
• 关键词: Extracellular Matrix; Tenascin-C; Tenascin-X; Tenascin-R; Gene Knockout; CpG island; ES cell; Chimeric Mouse

The tenascins are a growing family of extracellular matrix glycoproteins.To date three members of the family have been identified in mammals : tenascin-C (TN-C) (also called cytotactin), tenascin-R (TN-R) (for restrictin), and tenascin-X (TN-X).We have identified that TN-X gene is localized in the class III region of the major histocompatibility complex locus and the expression of TN-X is often reciprocal to that of TN-C in the developing mouse embryo.In order to test the possible involvement of TN-X in embryonic migrations and morphogenetic processes and in later events such as wound healing and oncogenesis, we have used homologous recombination in embryonic stem (ES) cells to inactivate the TN-X gene.Now we are attempting to generate strains of mice deficient in TN-X.Thus far we have constructed the targeting vectors which disrupt TN-X gene by deletion of the 5' portion removing the translation initiation codon.By using of the targeting vector, we have identified the two targeted clones among 1200 G418-resistant transfectants analyzed.To generate mice harboring the targeted TN-X allele, these targeted ES cell clones were injected into host 8-cell stage embryo.However no extensive ES-cell contribution was observed with the two clones as assayd by coat color chimerism, indicating that all chimeric embryos with high ES-cell contribution die between E13 and E16.The reason why these chimeric embryos die earlier in gestation remains to be determined.Further experiments should help to clarify this issue.As for the regulation of TN-X expression, Dr.Chiquet-Ehrismann (Friedrich Miescher Institute, Switzerland) has investigated the expression of TN-X in fibroblasts and carcinoma cells in culture.None of the growth factors or cytokines examined affected the expression level of TN-X,however, steroid hormone glucocorticoids, were found to downregulate TN-X mRNA levels and protein synthesis in fibroblasts but not carcinoma cells at physiological concentrations.

腱生蛋白是一个不断发展的细胞外基质糖蛋白家族。迄今为止，该家族的三个成员已在哺乳动物中被鉴定：腱生蛋白-C（TN-C）（也称为细胞增殖素），腱生蛋白-R（TN-R）（用于限制），TN-X基因定位于主要组织相容性复合体基因座的III类区域，TN-X基因的表达与TN-X基因的表达呈负相关，TN-X基因的表达与TN-X基因的表达呈负相关。在发育中的小鼠胚胎中，X通常与TN-C相反。为了测试TN-X在胚胎迁移和形态发生过程中以及在伤口愈合和肿瘤发生等后期事件中的可能参与，我们已经在胚胎干（ES）细胞中使用同源重组来扩增TN-目前，我们已经构建了通过删除翻译起始密码子的5'端部分来破坏TN-X基因的靶向载体，通过使用该靶向载体，我们在1200个G418抗性转染子中鉴定了两个靶向克隆。为了产生携带靶向TN-X等位基因的小鼠，将这些靶向ES细胞克隆注射到宿主8-细胞期胚胎中。然而，通过毛色嵌合性测定，这两个克隆没有观察到广泛的ES细胞贡献，提示所有ES细胞贡献高的嵌合体胚胎均在E13-E16之间死亡。这些嵌合体胚胎在妊娠早期死亡的原因尚待确定。进一步的实验将有助于阐明这一问题。至于TN-X表达的调控，Chiquet-Ehrismann博士（Friedrich Miescher Institute，瑞士）已经调查了TN-检测的生长因子或细胞因子均不影响TN-X的表达水平，然而，类固醇激素糖皮质激素，发现在生理浓度下下调成纤维细胞中TN-X mRNA水平和蛋白质合成，但不下调癌细胞中TN-X mRNA水平和蛋白质合成。

项目成果

T.Sakai, Y.Furukawa, R.Chiquet-Ehrismann, M.Nakamura, S.Kitagawa, T.Ikemura, and K.Matsumoto: "Tenascin-X Expression in Tumor Cells and Fibroblasts : Glucocorticoids as Potent Inhibitors in Fibroblasts" Journal of Cell Science. in press. (1996)

T.Sakai、Y.Furukawa、R.Chiquet-Ehrismann、M.Nakamura、S.Kitakawa、T.Ikemura 和 K.Matsumoto：“肿瘤细胞和成纤维细胞中的 Tenascin-X 表达：糖皮质激素作为成纤维细胞中的有效抑制剂”期刊

T.Sakai,Y.Furukawa,R.Chiquet-Ehrismann,M.Nakamura,S.Kitagawa,T.Ikemura,and K.Matumoto: "Tenascin-X Expression in Tumor Cells and Fibroblasts:Glucocorticoids as Potent Inhibitors in Fibroblasts" Journal of Cell Science. (印刷中). (1996)

T. Sakai、Y. Furukawa、R. Chiquet-Ehrismann、M. Nakamura、S. Kitakawa、T. Ikemura 和 K. Matumoto：“肿瘤细胞和成纤维细胞中的 Tenascin-X 表达：糖皮质激素作为成纤维细胞中的有效抑制剂”期刊细胞科学（正在出版）（1996）。