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

Abstract

Background: Diversity of immunoglobulins and the T cell antigen receptors is achieved via the recombination activating gene (RAG)-mediated rearrangement of variable (V), diversity (D) and joining (J) gene segments, and this underpins the efficient recognition of a seemingly limitless array of antigens. Analysis of V(D)J recombination activity is typically performed using extrachromosomal recombination substrates that are recovered from transfected cells and selected using bacterial transformation. We have developed a two-colour fluorescence-based system that simplifies detection of both deletion and inversion joining events mediated by RAG proteins.

Results: 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.

Conclusions: 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.

Figure 1

Physiological variable (V), diversity (D) and joining (J) recombination and analogous recombination substrates. (a) V and J segments on opposite strands (as found in the human Igκ locus) are joined by inversion between the recombination signal sequence (RSS)12 (filled triangle) and RSS23 (open triangle) motifs to generate a linked signal joint and coding joint (the VJ rearrangement). (b) V and J segments located on the same strand (as found in the human Igκ and λ loci) are recombined by deletion of the intervening DNA, leaving the coding segment on the chromosome and the signal joint on an excised circle of DNA. (c) In the inversion substrate, the DsRed gene and the EGFP gene are located on opposite stands, flanked by RSS12 and RSS23 motifs. V(D)J recombinase activity flips the segment allowing DsRed to be replaced by EGFP. (d) In the deletion substrate, the RSS motifs are in opposite orientations and flank the DsRed gene. On recombination, the DsRed gene is deleted, placing EGFP adjacent to the promoter. A single promoter is present in both constructs (curved arrow). The positions of the primer sequences F1 and R1, which were used to analyse recombination at the DNA level and for RT-PCR analysis, are shown.

Figure 2

Cell-type specificity of recombination. (a) Recombination activating gene (RAG)1 and RAG2 expression in pre-B 300-19P cells (REC+) and WEHI231 B cells (REC-) analysed by reverse transcription polymerase chain reaction (RT-PCR). Actin transcripts were detected in both cell lines. The RAG genes are intronless and assays were performed using reverse transcriptase (RT+) as well as in its absence (RT-) to ensure that signals were not derived from contaminating genomic DNA. (b) Detection of recombination by PCR (using the F1 and R1 primers shown in Figure 1). A PCR product of 155 bp was predicted following recombination. (c) RT-PCR to detect expression of EGFP mRNA from the transfected substrates. The PCR product diagnostic of EGFP transcripts from the recombined substrate is 155 bp. The 975-bp product detected from the deletion substrate transcripts represents a mRNA that traverses the entire DsRed gene and continues on through the EGFP gene. No products were detected in the absence of reverse transcriptase (- RT enzyme). Both DNA recombination and mRNA expression assays were performed from untransfected cells (Untrans) and in cells stably transfected with the deletion and inversion substrates (non-clonal populations grown under continuous selection) using 300-19P (REC+) and WEHI231 (REC-) cells. A (-) symbol indicates no added template.

Figure 3

Expression of the recombined substrates. (a) Fluorescence microscopy of EGFP and DsRed expression. The four panels show expression of the DsRed (from unrearranged template) and rearranged EGFP gene products from inversion and deletion substrates in both 300-19P and WEHI231 cells. (b) Flow cytometric analysis of 4-week cultures of untransfected and transfected recombinant 300-19P (REC+) and WEHI231 (REC-) cell lines. The fold increase in number of EGFP-expressing cells was established by assigning an arbitrary value of 1 for untransfected cells.