Cloning and analysis of the genes during the induction of mouse embryongenesis

小鼠胚胎发生诱导过程中基因的克隆与分析

• 批准号: 08457037
• 负责人: NAKANO Toru
• 金额: $ 4.74万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457037/
• 关键词: cDNA cloning; hematopoiesis; mice; secretory proteins

When mouse embryonic stem cells (ES cells) are co-cultured on macrophage colony stimulating factor deficient OP9 stromal cells, hematopoietic cells can be differentiated via mesodermal cells. In order to clone the genes of extraccellular signaling proteins functioning during this process, cDNA cloning was carried out by using the day 5 differentiation induced cells. We utilized Signal Sequence Trap (SST) method which is an efficient cloning strategy for secretary proteins and type I transmembrane proteins and the method was innovated by our group.As a result, one cDNA fragment which must contain authentic signal sequence. Full length cDNA was cloned by using the SST cDNA fragment as aprobe, this candidate gene turns out to be a murine homologue of the chicken gene whose expression is controlled by c-Myb but whose function is unknown yet. Now, the expression and the function of this gene is investigated.Meanwhile, we consider it possible to clone the genes involved in the induction of early embryogenesis of mice by examining the similarities and the differences of the gene expression between ES cells and primordial germ cells. And we start to collect mRNA from purified primordial germ cells. We are planning to carry out differential display or SAGE (serial analysis of gene expression) to clone the genes.

当小鼠胚胎干细胞（ES细胞）在巨噬细胞集落刺激因子缺陷的OP9基质细胞上共培养时，可以通过中胚层细胞分化造血细胞。为了克隆在此过程中起作用的细胞外信号蛋白的基因，使用第5天的分化诱导细胞进行cDNA克隆。我们利用信号序列陷阱（SST）方法，这是一种高效的分泌蛋白和I型跨膜蛋白克隆策略，该方法是我们课题组创新的。因此，获得了一个必须包含真实信号序列的cDNA片段。以SST cDNA片段为探针克隆全长cDNA，该候选基因是鸡基因的鼠同源物，其表达受c-Myb控制，但其功能尚不清楚。目前，我们对该基因的表达和功能进行了研究。同时，我们认为通过研究ES细胞和原始生殖细胞基因表达的异同，克隆参与小鼠早期胚胎发生诱导的基因是可能的。 。我们开始从纯化的原始生殖细胞中收集 mRNA。我们计划进行差异展示或SAGE（基因表达的系列分析）来克隆基因。

项目成果

T.Aoki, T.Nakano, et al: "Induction of Bip mRNA upon programmed cell death of differentiated PC12 cells as well as rat sympathetic neurons." J Biochem. 121. 122-127 (1997)

T.Aoki、T.Nakano 等人：“分化的 PC12 细胞以及大鼠交感神经元程序性细胞死亡时 Bip mRNA 的诱导”。

T.Aoki, T.Koike, T.Nakano, K.Shibahara, H.Nishimura, H.Kikuchi, T.Honjo: "Rat TAFII31 gene is induced upon programd cell death in differentiated PC12 cells deprived of NGF." BBRC. 234. 230-234 (1997)

T.Aoki、T.Koike、T.Nakano、K.Shibahara、H.Nishimura、H.Kikuchi、T.Honjo：“在缺乏 NGF 的分化 PC12 细胞中，大鼠 TAFII31 基因在程序性细胞死亡后被诱导。”

T.Aoki, T.Nakano, et al: "Rat TAFll31 gene is induced upon programmed cell death in differentiated PC12 cells deprived of NGF." BBRC. 234. 230-234 (1997)

T.Aoki、T.Nakano 等人：“在缺乏 NGF 的分化 PC12 细胞中，大鼠 TAFll31 基因在程序性细胞死亡时被诱导。”

J.L.de la Pompa, T.Nakano, et al: "Conservation of the Notch signalling pathway in mammalian neurogenesis." Development. 124. 1139-1148 (1997)

J.L.de la Pompa、T.Nakano 等人：“哺乳动物神经发生中 Notch 信号通路的保守”。

T.Aoki, T.Koike, T.Nakano, K.Shibahara, S.Kondo, H.Kikuchi, T.Honjo: "Induction of Bip mRNA upon programd cell death of differentiated PC12 cells as well as rat sympathetic neurons." J.Biochem.121. 122-127 (1997)

T.Aoki、T.Koike、T.Nakano、K.Shibahara、S.Kondo、H.Kikuchi、T.Honjo：“分化的 PC12 细胞以及大鼠交感神经元程序性细胞死亡时 Bip mRNA 的诱导”。