Isolation by fluorescence-activated cell sorting of cells of a human lymphoblastoid cell strain containing mutations in the lambda immunoglobulin gene.
Fluorescence-activated cell sorting (FACS) was used to establish clonal populations of a human lymphoblastoid cell strain that contain spontaneously occurring and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations in the lambda immunoglobulin gene. Multiple rounds of FACS using a monoclonal antibody specific for the membrane-expressed human lambda immunoglobulin were used to enrich the population fraction of cells lacking a wild-type lambda immunoglobulin on the cell surface. Approximately 20% of the clonal populations established after five rounds of FACS-mediated enrichment did not express the lambda immunoglublin epitope recognized by the monoclonal antibody used for selection. However, evaluation of the FACS-selected mutant clonal populations with a polyclonal antilambda antibody, or a monoclonal antibody directed against a different epitope on the lambda immunoglobulin made by the T5-1 cells, indicated that the mutant clonal populations expressed lambda immunoglobulin on their cell surfaces. Additionally, the presence of lambda mRNA and of both secreted and cytoplasmic lambda protein confirmed the transcription and translation of the lambda immunoglobulin gene. These data suggest that FACS-mediated selection employing epitope-specific monoclonal antibodies provides a powerful technique for isolation of cell populations that express mutations within the coding region of the lambda immunoglobulin gene.
McFarland
R D
RD
Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill 27599-7525.
Vincent
J L
JL
Smith
G J
GJ
D015152:Blotting, Northern; D002455:Cell Division; D002469:Cell Separation / Q000379:methods*; D002999:Clone Cells; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007146:Immunoglobulin lambda-Chains / Q000235:genetics*; D007700:Kinetics; D008214:Lymphocytes / Q000166:cytology* / Q000276:immunology; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D009154:Mutation
D007146:Immunoglobulin lambda-Chains; D008769:Methylnitronitrosoguanidine