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

Trancoso, Inês; Bonnet, Marie; Gardner, Rui; Carneiro, Jorge; Barreto, Vasco M.; Demengeot, Jocelyne; Sarmento, Leonor M.

Publication

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

2013-05-16Journal article

dc.contributor.author	Trancoso, Inês
dc.contributor.author	Bonnet, Marie
dc.contributor.author	Gardner, Rui
dc.contributor.author	Carneiro, Jorge
dc.contributor.author	Barreto, Vasco M.
dc.contributor.author	Demengeot, Jocelyne
dc.contributor.author	Sarmento, Leonor M.
dc.date.accessioned	2015-11-10T11:36:18Z
dc.date.available	2015-11-10T11:36:18Z
dc.date.issued	2013-05-16
dc.description.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.	pt_PT
dc.description.sponsorship	Portuguese League Against Cancer, Terry Fox Foundation, FCT fellowships,	pt_PT
dc.identifier	10.3389/fimmu.2013.00110
dc.identifier.citation	Trancoso I, Bonnet M, Gardner R, Carneiro J, Barreto VM, Demengeot J and Sarmento LM (2013) A novel quantitative fluorescent reporter assay for RAG targets and RAG activity. Front. Immunol. 4:110. doi: 10.3389/fimmu.2013.00110	pt_PT
dc.identifier.doi	10.3389/fimmu.2013.00110
dc.identifier.uri	http://hdl.handle.net/10400.7/485
dc.language.iso	eng	pt_PT
dc.peerreviewed	yes	pt_PT
dc.publisher	Frontiers Research Foundation	pt_PT
dc.relation.publisherversion	http://journal.frontiersin.org/article/10.3389/fimmu.2013.00110/abstract	pt_PT
dc.rights.uri	http://creativecommons.org/licenses/by/4.0/	pt_PT
dc.subject	recombination-activating gene 1	pt_PT
dc.subject	V(D)J recombination	pt_PT
dc.subject	green fluorescent proteins	pt_PT
dc.subject	reporter	pt_PT
dc.subject	inversion	pt_PT
dc.title	A Novel Quantitative Fluorescent Reporter Assay for RAG Targets and RAG Activity	pt_PT
dc.type	journal article
dspace.entity.type	Publication
oaire.awardURI	info:eu-repo/grantAgreement/FCT/3599-PPCDT/PTDC%2FBIA-BCM%2F108020%2F2008/PT
oaire.awardURI	info:eu-repo/grantAgreement/FCT/3599-PPCDT/PTDC%2FBIA-GEN%2F116830%2F2010/PT
oaire.citation.endPage	17	pt_PT
oaire.citation.startPage	1	pt_PT
oaire.citation.title	Frontiers in Immunology	pt_PT
oaire.citation.volume	4	pt_PT
oaire.fundingStream	3599-PPCDT
oaire.fundingStream	3599-PPCDT
project.funder.identifier	http://doi.org/10.13039/501100001871
project.funder.identifier	http://doi.org/10.13039/501100001871
project.funder.name	Fundação para a Ciência e a Tecnologia
project.funder.name	Fundação para a Ciência e a Tecnologia
rcaap.rights	openAccess	pt_PT
rcaap.type	article	pt_PT
relation.isProjectOfPublication	dad708a3-ee64-45ed-855c-0903018a6604
relation.isProjectOfPublication	210a45bb-0e47-4205-913a-112af5d6ea85
relation.isProjectOfPublication.latestForDiscovery	dad708a3-ee64-45ed-855c-0903018a6604