Development of new cloning method and its application for novel receptor identification in mast cells

新克隆方法的开发及其在肥大细胞新受体鉴定中的应用

• 批准号: 13670312
• 负责人: ONO Masao
• 金额: $ 2.18万
• 依托单位: Ehime 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-13670312/
• 关键词: Mast cell; Receptor; Expression cloning; Complementary DNA; Complement; 発現スクリーニング

Objectives of this study are 1) construction of mast cell cDNA library in retrovirus related vector (pMX), 2) development of new method for retrovirus-based expression cloning and 3) its application for identification of novel receptor associated with DAP12 activation subunit in mast cell. The results are summarized in the following three sections.1) Construction of mast cell cDNA libraryThe double stranded cDNA was prepared from bone marrow-derived IL-3-dependent murine mast cells by MMoLV-derived reverse transcriptase. The cDNA library was constructed in retrovirus-based plasmid vector (pMX) by ligation of cDNA and pMX (EcoRI and Not I restriction sites). The cDNA library was once amplified in E. coli (DH10B strain) by electro-transformation. After performing library constructions twice, we totally obtained a mast cell cDNA library containing more than 2 millions independent clones. Its integrity was monitored by PCR efficacy for the mRNA species rarely expressed in mast cell. We the … More reby concluded that our cDNA library was of good complexity to take advantage in further study.2) Indirect expression cloning method (mentioned as new method)We established a stable cell line (12Y5) expressing FLAG-tagged DAP12 protein in rat myeloma cell line by its relevant DNA transfection. When expressed the killer Ig-like receptor in 12Y5, which is known to associate with DAP12 and whose surface expression is dependent on association with DAP12, FLAG-tagged DAP12 was sorted to plasma membrane. On the other hand, when expressed the DAP12-independent receptor, FcgRIIb, FLAG-tagged DAP12 was not. These findings indicate that FLAG-tagged DAP12 is sorted to plasma membrane as depending on expression of DAP 12-associating receptor.3) Application of new method for identification of novel receptorWe applied the new method to identify novel receptor associating with DAP12 in mast cell. The mast cell cDNA library was introduced into 12Y5 by retrovirus-based method. After culturing for 2 days, cells positive for surface expression of FLAG were collected by FACS. Then the collected cells were expanded in culture for 1 week. This collection and expansion cycle was repeated three times. Finally, the FLAG-positive cells were cloned by limiting dilution method, each clone was subjected to sequence analysis. We isolated 4 independent clones through 4 times cloning performances. However no significant sequence was identified among them. As the main problem of this method, we concluded that false-positive clones could preferentially be amplified on the way of long-term culture for selection. Less

本研究的目的是1）在逆转录病毒相关载体（pMX）中构建肥大细胞cDNA文库，2）开发基于逆转录病毒的表达克隆新方法以及3）其在肥大细胞中与DAP12激活亚基相关的新型受体的鉴定中的应用。结果概括如下三部分：1)肥大细胞cDNA文库的构建利用MMoLV衍生的逆转录酶从骨髓来源的IL-3依赖性鼠肥大细胞制备双链cDNA。通过连接 cDNA 和 pMX（EcoRI 和 Not I 限制位点），在基于逆转录病毒的质粒载体 (pMX) 中构建 cDNA 文库。 cDNA文库通过电转化在大肠杆菌（DH10B菌株）中进行了扩增。经过两次建库，我们总共获得了包含超过200万个独立克隆的肥大细胞cDNA文库。通过对肥大细胞中很少表达的 mRNA 种类进行 PCR 功效来监测其完整性。我们得出的结论是，我们的cDNA文库具有良好的复杂性，可以在进一步的研究中发挥作用。2）间接表达克隆方法（称为新方法）我们通过相关DNA转染，在大鼠骨髓瘤细胞系中建立了表达FLAG标记的DAP12蛋白的稳定细胞系（12Y5）。当杀伤性 Ig 样受体在 12Y5 中表达时（已知该受体与 DAP12 相关且其表面表达依赖于与 DAP12 的相关性），带有 FLAG 标签的 DAP12 被分选至质膜。另一方面，当表达 DAP12 独立受体 FcgRIIb 时，带有 FLAG 标签的 DAP12 则不表达。这些发现表明，FLAG标记的DAP12根据DAP 12相关受体的表达被分选至质膜。3)应用新方法鉴定新受体我们应用新方法来鉴定肥大细胞中与DAP12相关的新受体。通过基于逆转录病毒的方法将肥大细胞cDNA文库导入12Y5。培养2天后，通过FACS收集FLAG表面表达阳性的细胞。然后将收集的细胞扩增培养1周。该收集和扩展循环重复三次。最后通过有限稀释法克隆FLAG阳性细胞，并对每个克隆进行序列分析。通过4次克隆实验，分离出4个独立克隆。然而，其中没有发现显着的序列。作为该方法的主要问题，我们得出的结论是，假阳性克隆可以通过长期培养的方式优先扩增以进行选择。较少的

项目成果

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Nakamura, A.、Ono, M.、Mukiwa, T. 和 Takai, T.：“（日本评论）Fcγ 受体缺陷型小鼠”Saishin-Igaku. 56. 51-56 (2001)

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藤野貴広, 小野栄夫: "Low-density lipoprotein receptor-related protein 5 (LRP5) is essential for normal cholesterol metabolism and glucose-induced insulin secretion"Proceeding National Academy of Science U. S. A. 100. 229-234 (2003)

Takahiro Fujino、Hideo Ono：“低密度脂蛋白受体相关蛋白 5 (LRP5) 对于正常胆固醇代谢和葡萄糖诱导的胰岛素分泌至关重要”，Proceeding National Academy of Science U. S. A. 100. 229-234 (2003)

Yuasa, T., Ono, M., and Takai, T.: "(Japanese review) Role of lyn kinase in mast cell activation and hypersensitivity"Rinsho-Meneki. 37. 567-572 (2002)

Yuasa, T.、Ono, M. 和 Takai, T.：“（日本评论）lyn 激酶在肥大细胞激活和超敏反应中的作用”Rinsho-Meneki。