iRAGu: A Novel Inducible and Reversible Mouse Model for Ubiquitous Recombinase Activity

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.

Keywords: 4-hydroxytamoxifen induction; V(D)J recombination; estrogen receptor; lymphocyte development; recombination activating gene; transgenic mouse model.

Figure 1

rag transgenes are transcribed in lymphoid and non-lymphoid tissues of Tg mice. rag1, rag2 and β-tubulin transcripts were detected by RT-PCR in thymus, bone marrow, liver, lung, kidney, and brain from one wild-type (WT) and two Tg endoR2^+/+ mice. Primers endo/tg detect both endogenous and transgenic rag transcripts. Results are representative of three independent experiments analyzing five different mice.

Figure 2

4-Hydroxytamoxifen (TAM) induction of Tg endoR2^−/− partially restores lymphocyte development. Young adult Tg-endoR2^−/− mice (Tg endoR2^−/−) were fed with normal (−) or TAM (+) food during 4 weeks before analysis. Untreated wild-type (WT) and recombination activating gene 2 (RAG2)-deficient (R2^−/−) mice served as control. (A) Representative FACS profiles of thymocytes, bone marrow, and lymph nodes cells. (B) Full analysis of thymocytes numbers and frequency of thymic DP (upper panels); frequency of E and F fractions and CD43⁺ B cells in the bone marrow (middle panels); concentration of seric Ig and lymphocyte numbers in lymph nodes (bottom panels). *, **, and *** stand for p-values <10⁻², 10⁻³, and 10⁻⁵, respectively. Results are representative of six independent experiments.

Figure 3

The T cell receptor (TCR) Jα repertoires of 4-hydroxytamoxifen (TAM)-induced Tg endoR2^−/− and wild-type (WT) mice are significantly different. Tg-endoR2^−/− mice (TgendoR2^−/−) were fed with normal TAM (+) food during 4 weeks before analysis. PCR and HTS sequencing of TCRα rearrangements were performed and represented as follow: each dot is a rearrangement, and the x-axis represent the distance between the Jα used in the rearrangement and the constant (C) region. The doted vertical bars represent the average distances to (C) of the Jα used in rearrangement in TAM-induced Tg endoR2^−/− (upper boxplot) and WT mice (bottom boxplot, from Mancini et al.). *** stands for a p-value <10⁻⁴.

Figure 4

Occurrence of Bcl11b cryptic recombination in 4-hydroxytamoxifen (TAM)-induced Tg endoR2^−/− animals. (A) Bcl11b locus, cryptic recombination sites and nested primers used in the fluctuation PCR analysis. Bcl11b exons are represented by red boxes, with introns represented as dashed lines connecting these boxes (note that bcl11b is on the reverse strand); cRSS are depicted as triangles within the introns, and primers used for the nested PCR are depicted by arrows. The bottom line shows an illegitimate Bcl11b recombination. (B) Fluctuation PCR analysis for each mouse concerned 24 reactions, each with 240 ng of genomic DNA, equivalent to a total of 0.96 million cells. Each row is the analysis of the total thymocytes of one Tg endoR2^−/− TAM⁺ mice. Recombination was detected in two out of three mice, at the frequency indicated in the right column.

Figure 5

Termination of 4-hydroxytamoxifen (TAM) administration prevents further lymphocyte development. (A,B) The upper lines represent the timelines of normal and TAM food administration in Tg endoR2^−/− animals, arrows indicate the time at analysis. (A) Administration of TAM during 2 weeks is sufficient to induce mature lymphocytes differentiation (left panels), while withdrawal of TAM for 4 weeks arrests lymphoid development. Representative FACS profiles of developing T cells in the thymus (upper panels) and B cells in the bone marrow (bottom panels). Data are representative of 3 independent experiments. (B) B cell development precedes T cells differentiation. Representative FACS profiles of B220⁺ peripheral blood mononuclear cell (PBMC, upper panels) and Thy1⁺ (lower panels) cells, after 1 week of TAM administration (D7) or followed by 1, 2, or 3 weeks of normal food. IgM⁺ and IgD⁺ B cells are observed from the first week of analysis, while only a few CD4⁺ cells are detected at that stage. Clear CD4⁺ and CD8⁺ T cells populations are detected at day 28. Data are representative of three independent experiments. (C) B cells consistently appear before T cells in peripheral blood. Kinetic analysis of B and T cells frequency in blood of Tg endoR2^−/− animals treated along the (B) regimen (N = 11 mice).

Figure 6

Alternated on and off 4-hydroxytamoxifen (TAM) administration reduces toxicity and promotes completion of the Vbeta repertoire. (A) Tg endoR2^−/− mice were treated for 16 weeks with alternate periods on (+) and off (−) TAM food (upper scheme), and body weight kinetics established along the treatments. (B) By week 16 of the alternate treatment in (A), thymocytes were analyzed by FACS, and wild-type (WT) thymocytes served as control. Shown is the proportion of Thy1⁺ thymocytes expressing each of the 8Vβ tested.

Figure 7

The Tg^hi line is leaky and exhibits impaired lymphocyte development/maintenance. Tg^hi endoR2^−/− and Tg^hi endoR2^+/− mice were induced (TAM+) or not (TAM−) for 4 weeks before analysis. Wild-type (WT) and Rag2^−/− mice served as control (A) Representative FACS analysis of thymocytes, bone marrow, and lymph nodes in Tg^hi mice. (B) Total thymocytes numbers. Tg^hi endoR2^−/− TAM-mice have higher number of thymocytes than R2^−/− mice, and Tg^hi endoR2^+/− mice exhibit a significant decrease of thymocytes when treated by TAM (*p = 0.016 and ****p < 0.0001 compared to untreated Tg^hi endoR2^+/− and WT mice, respectively). (C) Serum IgG concentration was assessed by quantitative enzyme-linked immunosorbent assay (ELISA). Tg^hi mice, whether endoR2^−/− or ^+/−, present significantly lower seric IgG levels than WT animals (*p = 0.03 untreated Tg^hi endoR2^−/−, and **p = 0.008 TAM⁺ Tg^hi endoR2^+/− mice, respectively). (D) Frequency of γδ T-cells in thymocytes was determined by FACS analysis. No statistical differences were found between the different groups. (E) Absolute number of mature T and B cells in pooled lymph nodes reveals severe lymphopenia in Tg^hi mice, irrespective of functional endoRag2 or TAM treatment. Results are representative of six independent experiments.

Figure 8

4-Hydroxytamoxifen (TAM)-induction of recombination activating gene (RAG) activity in non-lymphoid organs. (A,B) Mouse embryonic fibroblasts (MEF) cultures were established from Tg^hi and wild-type (WT) fetuses. (A) RT-PCR analysis revealed readily detectable Tg rag1and Tg rag2-ER transcripts in Tg^hi MEF. (B) RAG activity was assessed using the GFPi recombination assay. Upon infection with viral GFPi-mRFP particles, transgenic MEF were treated with either 2% ethanol (etOH) or 2% ethanol containing 200 nM of 4-hydroxy-tamoxifen (TAM) during 3 weeks. GFP expression revealed by FACS analysis accounts for induced RAG activity. Results are representative of two independent experiments. (C) The upper scheme represents alternated on and off TAM administration during the 49 weeks duration of the experiment. The graph shows survival of R2^−/− (gray dotted line) and Tg^hi (black line) mice reconstituted with R2^−/− bone marrow. Life span was not significantly different between the groups. N = 6 for each group.