title: Retroviral transduction of lineage antigen-negative ((Lin−) cells: a valuable alternative for the generation of T cell receptor (TCR) retrogenic mice.
abstract: Mice with virtually all T cells expressing a single T cell receptor (TCR) on their surface have been instrumental in understanding the development of immature thymocytes. For many years, such an engineering has been achieved essentially by inserting rearranged TCR α and β chain coding sequences into the genome through co-microinjection into fertilized eggs (TCR transgenesis). More recently, a novel methodology relying on the reconstitution of T cell deficient hosts with retrovirally-transduced multipotent bone marrow cells has been developed. Hence, TCR retrogenesis allows for the in vivo study of given TCR specificities in a faster and less expensive manner. While initial procedures were taking advantage of 5-Fluorouracil (5-FU) treatment of RAG-deficient or SCID donor mice as source of haematopoietic stem cells, we used bone marrow cell suspensions enriched in lineage antigen-negative (Lin−) cells from untreated donors for TCR retrogenesis. In contrast to cells from 5-FU-treated donors, transduced Lin−cells consistently generated a sizable retrogenic pool of thymocytes and required less donor mice. In such retrogenic mice, immature thymocytes bearing a major histocompatibility complex (MHC) class II-restricted TCR differentiated into the expected CD4 mature T cell lineage and populated the peripheral lymphoid organs where they retained the capacity to react to their cognate ligand. Lin− cell-enriched BM cells represent therefore, a reliable alternative to 5-FU treatment for retroviral transduction of haematopoietic stem cells and TCR retrogenic derivation.
authors.affiliation: Centre de Physiopathologie de Toulouse Purpan, Université Toulouse III Paul-Sabatier, Toulouse, F-31300, France. christophe.viret@inserm.fr
authors.forename: Christophe
authors.initials: C
authors.lastname: Viret
authors.forename: Charlotte
authors.initials: C
authors.lastname: Cresson
authors.forename: Cyril
authors.initials: C
authors.lastname: Broccardo
authors.forename: Sylvie
authors.initials: S
authors.lastname: Guerder
mesh_terms: D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000276:immunology*; D019070:Cell Lineage / Q000235:genetics / Q000276:immunology; D005434:Flow Cytometry; D005472:Fluorouracil / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008198:Lymph Nodes / Q000166:cytology / Q000276:immunology; D051379:Mice; D057507:Mice, 129 Strain; D016688:Mice, Inbred NOD; D018345:Mice, Knockout; D016513:Mice, SCID; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology*; D012190:Retroviridae / Q000235:genetics; D013154:Spleen / Q000166:cytology / Q000276:immunology; D060168:Thymocytes / Q000276:immunology / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D000963:Antimetabolites; D011948:Receptors, Antigen, T-Cell; D005472:Fluorouracil