Practical Improvement of Signal Sequence Trap Method

信号序列陷阱法的实用改进

• 批准号: 07557330
• 负责人: NAKANO Toru
• 金额: $ 1.47万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557330/
• 关键词: cDNA cloning; hematopoiesis; mice; secretory proteins; サイトカイン; 造血

Signal Sequence Trap (SST) method is an efficient cloning strategy for secretary proteins and type I transmembrane proteins and the method was innovated by our group. An independent research group of Genentech, a venture company in USA,reported a novel method by using secretion of yeast invertase out to the cells based on our report (Klein, RD,et al.Proc Natl Acad.Sci.U.S.A.1996,93 (14) : 7108). Comparing our original SST method and Genentech's novel method, the novel method turns out to be far more effective than our original method by a magnitude of 10,000. Then, we consider it reasonable to adapt the new method, instead of improving our original method. We start a large scale screening by using the novel yeast method.Based on the cDNA fragment obtained by SST method, full length cDNA's were cloned. Next, gene products of the cDNA's were obtained by expressing the cDNA's in Cos cells. We analyzed whether those proteins obtain some effects on the development and differentiation of blood cells. For this purpose, we utilized in vitro differentiation induction from mouse embryonic stem cells on OP9 stromal cells (OP9 system). This analysis is under way, however, no cDNA's containing significant effects on the development and differentiation of blood cells have been obtained.

信号序列捕获法（SignalSequenceTrap，SST）是本课题组创新的一种高效的分泌蛋白和I型跨膜蛋白克隆方法。美国风险投资公司Genentech的一个独立研究小组基于我们的报告报道了一种利用酵母转化酶分泌到细胞中的新方法（Klein，RD，et al.Proc Natl Acad.Sci.U.S.A.1996，93（14）：7108）。将我们原来的SST方法和Genentech的新方法进行比较，新方法比我们原来的方法有效10，000个数量级。然后，我们认为采用新的方法，而不是改进我们的原始方法是合理的。我们采用酵母法进行了大规模筛选，并以SST法获得的cDNA片段为基础，克隆了全长cDNA。接着，通过在Cos细胞中表达cDNA获得cDNA的基因产物。我们分析了这些蛋白质是否对血细胞的发育和分化有影响。为此，我们利用小鼠胚胎干细胞在OP9基质细胞上的体外分化诱导（OP9系统）。该分析正在进行中，然而，尚未获得对血细胞的发育和分化具有显著影响的cDNA。

项目成果

T.Aoki, T.Nakano, et al: "Rat TAFII31 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 TAFII31 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.Era, T.Nakano, et al: "Thrombopoietin enhances proliferation and differentiation of murine yolk sac erythroid progenitors." Blood. 89. 1207-1213 (1997)

T.Era、T.Nakano 等人：“血小板生成素增强小鼠卵黄囊红系祖细胞的增殖和分化。”

H.Nishimura et al.: "Developmentally regulated expression of the PD-1 protein on the surface of double negative (CD4^-,CD8^-) thymocytes." Int.Immunol.(in press).

H.Nishimura 等人：“PD-1 蛋白在双阴性 (CD4^-,CD8^-) 胸腺细胞表面的发育调节表达。”

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”。