Tese de mestrado, Biologia (Biologia Molecular Humana), 2009, Universidade de Lisboa, Faculdade de CiênciasResumo alargado em português disponível no documentoRecombination-Activating Genes (RAG) 1 and 2, form the site specific recombinase that mediates V(D)J recombination at the antigen receptor loci, a process responsible for lymphocyte diversity. RAG can also interact with degenerated recognition signal sequences (cRSSs) distributed throughout the genome and potentially induce genomic instability. To this date, no available reporter of RAG activity has gathered the versatile features of a molecular tool with a simple method of readout. Our laboratory has recently generated GFPi, a novel episomal/retroviral fluorescence-based molecular tool that allows for fast assessment of RAG activity in various cell types. We have found that the GFPi reporter can not only provide in vitro quantitative measurement transiently, being sensitive to RSS sequence degeneration, but also ex vivo in its integrated form as a stable substrate. We have also optimized the in vitro recombination assay (IVRA) for the detection of a broader window of RAG activity values. This novel assay allowed us to quantify the efficiency of RAG recombination of a selected set of cRSSs, namely a previously undetected cRSS found in the first exon of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) gene, which is known to be involved in leukemogenesis. PTEN cRSS was proved to be as functional as other V(D)J loci RSSs, and this activity correlates with a novel mutation which was recently found in a leukaemic patient in this same site. We also made use of the retroviral-based form of GFPi in order to quantify endogenous RAG activity in human haematopoietic tumour cell lines of lymphoid and myeloid origin. Although we have found lineage-specific differences on RAG activity (namely in Reh B-cell line which presented intense RAG activity), we found no signs of promiscuous endogenous RAG activity in myeloid cell lines in a short timespan. Thus, we believe the GFPi reporter gathers all the requisites for an efficient RAG activity reporter, coupled with a fast and direct method of readout. Moreover, we now possess a robust tool that is suitable for a number of applications, namely unravelling the relation between RAG promiscuous activity and leukaemogenesis, which was preliminary addressed in this work
