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

众所周知，B淋巴细胞产生的抗体在体液免疫中起着不可低估的作用。这些抗体具有广泛的抗原特异性，是由重组激活基因(RAG)-1和RGA-2催化的Ig基因重排产生的。Almough说，目前还没有一种简便的方法来检测重排B细胞的祖细胞。为了解决这一问题，我们试图建立检测免疫球蛋白基因重排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两个RAG识别序列之间。将此载体逆转录病毒导入CHO细胞，只观察到DsRED的红色荧光。另一方面，当RAG-1和RAG-2基因共转染这些红色CHO细胞时，部分细胞呈绿色荧光阳性。这些结果表明，整合的逆转录病毒载体被RAG重排，DsRED和EGFP基因的方向发生了逆转。此外，当胸腺切片在含有逆转录病毒的培养液中培养时，在RAG阳性的未成熟T细胞中观察到绿色荧光，结合这些结果，证明了逆转录病毒介导的荧光标记对RAG重排的B细胞和T细胞的检测和分析是有用的。

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.