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

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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.