Detecting variable (V), diversity (D) and joining (J) gene segment recombination using a two-colour fluorescence system

This system employs two fluorescent reporter genes that differentially mark unrearranged substrates and those that have undergone RAG-mediated deletion or inversion events. The recombination products bear the hallmarks of true V(D)J recombination and activity can be detected using fluorescence microscopy or flow cytometry. Recombination events can be detected without the need for cytotoxic selection of recombination products and the system allows analysis of recombination activity using substrates integrated into the genome.This system will be useful in the analysis and exploitation of the V(D)J recombination machinery and suggests that similar approaches could be used to replace expression of one gene with another during lymphocyte development.The antigen receptor loci of B and T lymphocytes exhibit a unique mechanism of control amongst the genes of multicellular organisms. The production of functional immunoglobulin (Ig) and T cell receptor (TCR) genes is accomplished through a tightly regulated process of recombination. Variable (V), diversity (D) and joining (J) gene segments of antigen receptor loci are assembled into a functional coding unit by a series of site-specific recombination events mediated by the products of recombination activating gene (RAG)1 and RAG2 [1]. Recombination is targeted to specific sites by the recombination signal sequences (RSS), which flank the gene segments. RSS motifs consist of a conserved heptamer (CACAGTG) separated from a conserved nonamer (ACAAAAACC) by a spacer of variable sequence of either 12 or 23 base pairs (bp). Recombination occurs between an RSS with a 12-bp spacer (RSS12) and an RSS with a 23-bp spacer (RSS23) and the intervening DNA is either deleted or inverted depending upon the orientation of the two signals (Figure 1). Double strand breaks introduced at the RSS motifs by the RAG proteins are then resolved by non-homologous end joining. Two products are generated, a signal joint in which the RSS motifs are joined