Abstract
Developing lymphocytes express the recombination activating genes (RAGs) 1 and 2 products that form a site specific recombinase complex (RAG), introducing double strand DNA breaks (DSBs) at recombination signal sequences (RSSs) flanking the V, D, and J gene segments in the antigen receptor loci. The subsequent steps in the reaction consist in the ligation of DSBs by ubiquitous enzymes of the non-homologous end joining DNA repair pathway. This mutagenesis process is responsible for the generation of the very large clonal diversity of T and B lymphocytes, itself allowing the recognition of a virtually open-ended antigenic universe. Sequences resembling RSS are found at high frequency all over the genome, and involved in RAG mediated illegitimate recombination and translocations. Hence, natural and induced ectopic activity of RAG is a threat to the genome only recently underscored. Here, we report and characterize a novel mouse transgenic system for which ubiquitous expression of the recombinase is inducible. In this system, the RAG1 protein is constitutively expressed and functional, while the RAG2 protein, coupled to the estrogen receptor, becomes functionally active upon 4-hydroxytamoxifen (TAM) administration. We describe two transgenic lines. The first one, when introgressed into an endogenous Rag2−/− genetic background is faithfully recapitulating lymphocyte development, repertoire dynamics and cryptic rearrangements, in a TAM-dependent manner. In this model, deprivation of TAM is followed by lymphocyte development arrest, evidencing the reversibility of the system. The second transgenic line is leaky, as the transgenes promote lymphocyte differentiation in absence of TAM treatment. Upon TAM-induction defects in lymphocytes composition and global health reveals the deleterious effect of uncontrolled RAG activity. Overall, this novel transgenic model provides a tool where RAG activity can be specifically manipulated to assess the dynamics of lymphocyte differentiation and the challenges imposed by the recombinase on the vertebrate genome.
Highlights
B and T lymphocytes are the prerogative of the adaptive immune system thanks to their ability to generate a quasi-infinite diversity of antigen receptors through a mechanism called V(D)J recombination
The recombinase complex is formed by the two lymphoidspecific recombination activating genes (RAG1 and 2) proteins, which are sufficient to induce a double strand DNA break, provided the gene segments are flanked by functional recombination signal sequences [RSSs; [4]], which are the specific target sequences of the recombinase
Using transgenic mice crossed on the recombination activating gene 2 (RAG2)-deficient background, we show that the rag1 and rag2-estrogen receptor (ER) transgenes are ubiquitously expressed, that their activity rescues the development of B and T lymphocytes upon TAM-administration, and that this induction is reversible
Summary
B and T lymphocytes are the prerogative of the adaptive immune system thanks to their ability to generate a quasi-infinite diversity of antigen receptors through a mechanism called V(D)J recombination. This is how the TCRβ locus complete recombination occurs in T cells only, before recombination of the TCRα locus and that a single functional TCRβ is produced per cell, which is called allelic exclusion [24] This last regulation level is driven by changes in chromatin accessibility of the antigen receptor loci that become accessible to the recombinase at the right juncture, to ensure proper lymphocyte development [23]. To assess the significance of the strict tissue and developmental stage specificities of the rag genes expression, we had previously generated transgenic mice in which both rag genes were constitutively coexpressed [26] and we reported a premature death of transgenic animals before 4 weeks of age, which was associated with severe lymphopenia To overcome this development constrain, we developed a new inducible model for ubiquitous expression of the recombinase. We describe another line of transgenic mice that is leaky, and which exhibits defects of lymphocyte development
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