A Novel Quantitative Fluorescent Reporter Assay for RAG Targets and RAG Activity

Abstract

Recombination-Activating Genes (RAG) 1 and 2 form the site specific recombinase that mediates V(D)J recombination, a process of DNA editing required for lymphocyte development and responsible for their diverse repertoire of antigen receptors. Mistargeted RAG activity associates with genome alteration and is responsible for various lymphoid tumors. Moreover several non-lymphoid tumors express RAG ectopically. A practical and powerful tool to perform quantitative assessment of RAG activity and to score putative RAG-Recognition signal sequences (RSS) is required in the fields of immunology, oncology, gene therapy, and development. Here we report the detailed characterization of a novel fluorescence-based reporter of RAG activity, named GFPi, a tool that allows measuring recombination efficiency (RE) by simple flow cytometry analysis. GFPi can be produced both as a plasmid for transient transfection experiments in cell lines or as a retrovirus for stable integration in the genome, thus supporting ex vivo and in vivo studies. The GFPi assay faithfully quantified endogenous and ectopic RAG activity as tested in genetically modified fibroblasts, tumor derived cell lines, developing pre-B cells, and hematopoietic cells. The GFPi assay also successfully ranked the RE of various RSS pairs, including bona fide RSS associated with V(D)J segments, artificial consensus sequences modified or not at specific nucleotides known to affect their efficiencies, or cryptic RSS involved in RAG-dependent activation of oncogenes. Our work validates the GFPi reporter as a practical quantitative tool for the study of RAG activity and RSS efficiencies. It should turn useful for the study of RAG-mediated V(D)J and aberrant rearrangements, lineage commitment, and vertebrate evolution.

NF-κB and AKT signaling prevent DNA damage in transformed pre-B cells by suppressing RAG1/2 expression and activity

“…25 Abltransformed pre-B cells can be induced to undergo differentiation toward small pre-B cells with the Abl kinase inhibitor STI571. Figure 2B).…”

“…25 The retroviral RAG-reporter and IkBaSR (IkBa S32A/S36A) constructs were cotransfected with the pCL-ECO plasmid in Phoenix-A cells. For retroviral transduction, cells and retroviral supernatants were spun for 90 minutes at 2000g onto retronectincoated 24-well plates (5 3 10 5 cells per well) according to the manufacturer's instructions (Takara Bio Inc., Otsu, Shiga, Japan).…”

PTEN microdeletions in T-cell acute lymphoblastic leukemia are caused by illegitimate RAG-mediated recombination events

“…28 Normal thymocytes were isolated from thymic tissue obtained from children undergoing cardiac surgery. 24,29 Informed consents were in accordance with the institutional review boards of the Erasmus MC (Rotterdam, The Netherlands), the ethics committee of the City of Hamburg, Germany, the Hospital Sta. Cruz, Centro Hospitalar de Lisboa Ocidental (Lisboa, Portugal), and the Declaration of Helsinki.…”

“…This latter is also supported by in vitro recombination assays using T-ALL cell lines, demonstrating that about 1% of leukemic cells or less will undergo recombinations of the reporter construct within a 1-week time frame. 29 Because intraclonal heterogeneity at diagnosis and clonal evolution at relapse are known to occur in ALL, 46,[61][62][63][64] we checked whether PTEN microdeletions in minor leukemic clones at presentation of disease become clonally selected following xenograft transplantation just like lentiviral PTEN-silenced T-ALL blasts. 34 However, we did not observe preferential selection of leukemic cells with PTEN microdeletions to near clonal levels following xenotransplantation.…”

“…We used the GFPi-mRFP RAG reporter construct 29 ( Figure 3A), in which the 12-spacer cRSS1 was cloned in combination with a consensus 23-spacer RSS. Also, the 23-spacer RSSs (cRRS2, cRSS3, and cRRS4) were cloned in combination with a consensus 12-spacer RSS.…”