Visualization of alteration of antigen specificity in mature B lymphocytes by multiple fluorescent labelling

通过多重荧光标记可视化成熟 B 淋巴细胞中抗原特异性的改变

基本信息

批准号：
12650789
负责人：
HIKIDA Masaki
金额：
$ 0.77万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12650789/
关键词：
GFP DsRED RAG retrovirus 蛍光タンパク遺伝子導入抗体遺伝子 Bリンパ球

项目摘要

It is well known that antibodies produced by B lymphocytes play indespensable role in humroal immunity. These antibodies possess wide variety of antigen-sepcificity, which was generated by Ig gene rearrangement catalized by recombination activating gene (RAG)-1 and RGA-2. Almough, there is no convenient methodology for detecting the progenitor of rearranged B cells. In order to solve this issue, we tried to establish novel method for detection of Ig gene rearranged B cells.Two genes of fluorescent protein DsRED and EGFP were ligated in the opposite orientation between 2 RAG recognition sequences. When this construct was transfected to CHO cells retrovirally, only the red fluorescence of DsRED was observed. On the other hand when RAG-1 and RAG-2 genes were cotransfected to these red CHO cells, some of these cells became EGFP positive and green fluoresccence was observed. These result indicate that integrated retroviral vector was rearranged by RAG and the orentation of DsRED and EGFP genes were reverted. Furthermore, when thymus sections were cultured in the medium which contains retrovirus green fluorescence was observed in RAG positive immature T progenitors.Together with these results, it is demonstrated that retrovirus-mediated fluorescent labelling is useful for the detection and analysis of RAG rearranged B and T cells.

众所周知，B淋巴细胞产生的抗体在体液免疫中发挥着不可或缺的作用。这些抗体具有多种抗原特异性，是由重组激活基因（RAG）-1和RGA-2催化的Ig基因重排产生的。然而，目前还没有方便的方法来检测重排 B 细胞的祖细胞。为了解决这个问题，我们试图建立检测Ig基因重排的B细胞的新方法。将荧光蛋白DsRED和EGFP的两个基因以相反的方向连接在2个RAG识别序列之间。当将该构建体逆转录病毒转染至 CHO 细胞时，仅观察到 DsRED 的红色荧光。另一方面，当RAG-1和RAG-2基因共转染到这些红色CHO细胞中时，其中一些细胞变成EGFP阳性并且观察到绿色荧光。这些结果表明整合的逆转录病毒载体被RAG重排并且DsRED和EGFP基因的方向被恢复。此外，当胸腺切片在含有逆转录病毒的培养基中培养时，在RAG阳性未成熟T祖细胞中观察到绿色荧光。与这些结果一起，证明逆转录病毒介导的荧光标记可用于检测和分析RAG重排B和T 细胞。