Analysis of transgenic mice expressing the recombination activating genes 1 and 2

The Recombination Activating Gene products (RAG1 and RAG2) are site-specific DNA recombinases essential for the diversification of Immunoglobulin and T-Cell Receptor loci in jawed vertebrates. Expression of both genes is lymphocyte specific and tightly regulated throughout lymphopoiesis and cell cycle. Both RAG1 and RAG2 deficient animals are devoid of mature B- and T-lymphocytes. In the frame of a project that aim at establishing the contribution of RAG1 and RAG2 genes in vertebrate genome instability, the laboratory had produced mice transgenic for either the RAG1 or the RAG2 gene. The transgenic constructs contain the MHC class I promoter, the Eµ enhancer, either RAG1 or RAG2 coding sequences and the beta-globin termination of transcription signal, to allows ubiquitous over-expression of either RAG1 or RAG2 proteins. In this work, we analyzed three RAG1 and two RAG2 transgenic mouse lines, with the objective of identifying the mice that express ubiquitously a functional transgene. The conduction of this work implied the follow-up of several breedings and the maintenance of each line in a stable colony. The breeding performance and the transgene transmission pattern were recorded, revealing heterogeneity among the five lines. Ubiquitous expression of the RAG genes was confirmed by testing lymphoid and non-lymphoid tissues for the presence of transgene specific mRNAs, revealing again heterogeneity among the five lines. The functionality of the integrated transgene in each line was tested by genetic complementation. To this aim RAG1 and RAG2 transgenic animals were bred with mice bearing a null mutation for the respective endogenous RAG gene (RAG1 or RAG2 knock-out mice, respectively). Backcrossing the transgenic F1 animals with mutant mice allowed the selection of transgenic progenies homozygote for the mutation. These animals served to test the functional complementation of the mutation by the transgene. In a first screen, peripheral blood monocytes were tested by FACS analysis for the presence of B- and T-lymphocytes. This measurement indicated that only one RAG1 and one RAG2 transgenic line carried a functional transgene able to partially rescue lymphopoiesis. Further analysis of the number and nature of the lymphocyte subsets in the primary and peripheral lymphoid organs confirmed accumulation of precursors and production of limited number of mature B- and T-cells. Strikingly, despite obvious lymphopenia, serum immunoglobulin levels were elevated. Moreover, these animals displayed alopecia and colitis, evoking pathologies associated with deregulated lymphocyte activation. The finding that only two out of the five mouse lines we analyzed carried a functional transgene, together with earlier studies in the laboratory revealing that two RAG2 transgenic founders died prematurely as well as their progeny, strongly suggest that the transgenes are toxic, at some level that remain to be defined. Consistent with this hypothesis, we show that B- and T-lymphocyte progenitor numbers are reduced by half in RAG competent animals when these carry the functional RAG1 and RAG2 transgenes. Finally, lymphopenia due to impaired RAG activity associates with various autoimmune and immunopathologies defining the Omenn syndrome in humans. Our analyses suggest that mice transgenic for the RAG gene (on an endogenous RAG KO genetic background) may serve as an experimental model for this rare but dramatic condition. Future work will formally test this hypothesis.