Novel expression cloning methods based on retrovirus mediated gene transfer.

基于逆转录病毒介导的基因转移的新型表达克隆方法。

• 批准号: 10559006
• 负责人: KITAMURA Toshio
• 金额: $ 8.26万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10559006/
• 关键词: retrovirus; expression cloning; cDNA library; signal sequence trap; GFP; サイトカイン; サイトカインレセプター; TNFレセプター; 転写因子

We previously established a high-efficiency retrovirus-mediated expression cloning method. Using this system, we have now developed a novel expression cloning method in which cDNAs can be isolated based on the subcellular localization of their products. We make cDNAs using random hexamer and fuse them to 5' endo of the cDNA of green fluorescent protein(GFP)in the pMX retrovirus vector to construct a cDNA-GFP fusion retrovirus library. The derived retroviruses are then infected to NIH3T3 cells to identify a cDNA of interest based on the subcellular localization of its protein product such as nucleus, nucleoli, Golgi apparatus, and cell surface. Using this strategy, we have identified a novel zinc finger protein from fetal mouse liver cells that contain hematopoietic progenitor cells.We have also established a novel signal sequence trap method which can identify cDNA fragments containing signal sequence that encode secreted and cell-surface. Signal sequence trap was originally developed in Kyoto University, and was improved by a group in Genentech, isolate cDNAs with signal sequences that encode such proteins have been established. In our method termed, cDNA fragments fused to an extracellular deletion mutant of the constitutively active receptor for thrombopoietin(MPL)were introduced into IL-3-dependent cells via retrovirus infection followed by the selection of factor-independent clones. Our method is much quicker and more accurate than the previously published methods. We have identified three novel receptors(a type I cytokine receptor, a TNF receptor-like molecule, and a novel low-density lipoprotein receptor-related protein)and several novel secreted molecules by SST-REX, and are now characterizing these molecules using various strategies including knockout mice.

我们先前建立了一种高效逆转录病毒介导的表达克隆方法。使用该系统，我们现在开发了一种新型的表达克隆方法，可以根据其产品的亚细胞定位来隔离cDNA。我们使用随机六聚体制作cDNA，并将它们融合到PMX逆转录病毒载体中绿色荧光蛋白（GFP）的5'内托中，以构建cDNA-GFP融合逆转录病毒库。然后将衍生的逆转录病毒感染到NIH3T3细胞中，以基于其蛋白质产物的亚细胞定位（例如核，核仁，高尔基体设备和细胞表面）鉴定感兴趣的cDNA。使用这种策略，我们已经确定了一种来自胎儿小鼠肝细胞的新型锌指蛋白，该锌蛋白包含造血祖细胞细胞。我们还建立了一种新型的信号序列陷阱方法，该方法可以识别包含编码分泌和细胞表面的信号序列的cDNA片段。信号序列陷阱最初是在京都大学开发的，并由Genentech的一组分离cDNA进行了改进，该cDNA具有编码此类蛋白质的信号序列。在我们所谓的方法中，通过逆转录病毒感染引入了组成型活性受体（MPL）的组成型活性受体（MPL）的细胞外缺失突变体的cDNA片段。我们的方法比以前发布的方法更快，更准确。我们已经确定了三个新型受体（一种I型细胞因子受体，TNF受体样分子和一种新型的低密度脂蛋白受体相关蛋白）和SST-Rex的几种新型分泌分子，现在正在使用包括敲除小鼠在内的各种策略来表征这些分子。

项目成果

Sugiyama, T., Kumagai, H., Morikawa, Y., Wada, Y., Sugiyama, A., Yasuda, K., Yokoi, N., Kojima, T., Nosaka, T., Senba, E., Kimura, S., Kadowaki, T., Kodama, T., and Kitamura, T.: "A novel low-density lipoprotein receptor-related protein mediating cellular

杉山，T.，熊谷，H.，森川，Y.，和田，Y.，杉山，A.，安田，K.，横井，N.，小岛，T.，野坂，T.，森场，E.，

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Kitamura,T.：“通过逆转录病毒介导的表达克隆分离 T 细胞抗原的 cDNA。”

Kawashima,T.: "Interleukin-12 induces tyrosine phosphorylation of an 85-kDa protein associated with the interleukin-12 receptor β1 subunit." Cell.Immunol.186. 39-44 (1998)

Kawashima, T.：“Interleukin-12 诱导与 IL-12 受体 β1 亚基相关的 85 kDa 蛋白的酪氨酸磷酸化。Cell.Immunol.186 (1998)。

Kawashima, T., Hirose, K., Satoh, T., Ikeda, Y., Kaziro, Y., Nosaka, T.and Kitamura, T.: "MgcRacGAP is involved in the control of growth and differentiation of hematopoietic cells."Blood. 96. 2116-2124 (2000)

Kawashima, T.、Hirose, K.、Satoh, T.、Ikeda, Y.、Kaziro, Y.、Nosaka, T. 和 Kitamura, T.：“MgcRacGAP 参与造血细胞生长和分化的控制。

Kitamura,T.: "Isolation of cDNAs for T cell antigens by retrovirs-mediated expression cloning"T Cell Protocols,Methods in Molecular Biology.. 143-152 (2000)

Kitamura,T.：“通过逆转录病毒介导的表达克隆分离 T 细胞抗原的 cDNA”T 细胞方案，分子生物学方法.. 143-152 (2000)