Rapid in vivo analysis of mutant forms of the LAT adaptor using Pax5-Lat double-deficient pro-B cells.
Following injection into recombinase-activating gene-deficient (Rag1(-/-)) mice, pro-B cells lacking the Pax5 transcription factor (Pax5(-/-)) develop into most major hematopoietic lineages, with the notable exception of B cells. We assessed whether Pax5(-/-) pro-B cells that were also rendered deficient for the linker for activation of T cells (LAT), an adaptor essential for T cell receptor signaling, can be used for the rapid in vivo analysis of mutant forms of LAT. We showed that Pax5(-/-) Lat(-/-) pro-B cell lines can be infected with recombinant retroviruses expressing a LAT cDNA and sorted for the expression of LAT. When injected into Rag1(-/-) mice, they restore normal intrathymic T cell development and give rise to functional peripheral T cells. Considering that the handling of Pax5(-/-) pro-B cell lines is easier than that of bone marrow hematopoietic precursors, we used them for the rapid functional analysis of a novel Lat allelic series. When compared to knock-in and transgenic approaches, a major advantage of our Pax5(-/-) pro-B cell-based experimental approach consists in the production of mice bearing a given mutation within 2-3 months. Therefore, it constitutes a powerful first-line screen for mutations worth fastidious knock-in approaches.
Ardouin
Laurence
L
Centre d'Immunologie de Marseille-Luminy, INSERM, CNRS, Université de la Méditerranée, Marseille, France.
Rolink
Antonius G
AG
Mura
Anne-Marie
AM
Gommeaux
Julien
J
Melchers
Fritz
F
Busslinger
Meinrad
M
Malissen
Marie
M
Malissen
Bernard
B
D048868:Adaptor Proteins, Signal Transducing / Q000172:deficiency / Q000235:genetics* / Q000276:immunology; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D002454:Cell Differentiation / Q000276:immunology; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000172:deficiency* / Q000235:genetics / Q000276:immunology; D005434:Flow Cytometry; D005821:Genetic Techniques; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008817:Mice, Mutant Strains; D009154:Mutation; D051757:PAX5 Transcription Factor; D010750:Phosphoproteins / Q000172:deficiency / Q000235:genetics* / Q000276:immunology; D013234:Stem Cells / Q000166:cytology / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D014157:Transcription Factors / Q000172:deficiency* / Q000235:genetics / Q000276:immunology
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C491734:Lat protein, mouse; D008565:Membrane Proteins; D051757:PAX5 Transcription Factor; C497282:Pax5 protein, mouse; D010750:Phosphoproteins; C493780:Slc50a1 protein, mouse; D014157:Transcription Factors