Study of T-cell signaling by somatic cell mutagenesis and complementation cloning.
Somatic cell mutagenesis is a powerful genetic approach used in dissection of signal transduction pathways in mammalian cells. Here we describe a method that has been successfully used to identify and analyze components of the NF-kappaB signaling pathway in Jurkat T cells using the combination of somatic cell mutagenesis and a complementation cloning strategy. By treating Jurkat T cells with the chemical mutagen ICR191, mutant T-cell lines can be selected that have a deficiency in a given biological response or in the expression of a particular selectable marker. Mutant phenotypes can be rescued by retroviral-mediated delivery of cDNA libraries and subsequent selection of rescued cell clones by flow cytometric cell sorting. Cell lines reverting back to the wild-type phenotype due to the ectopic expression of the exogenous gene can then be evaluated by functional assays. The gene rendering the reversion of the mutant phenotype may be isolated by PCR using library vector-specific primers. Clearly, creation of a somatic cell genetic system can yield exciting new advances in deciphering signal transduction events by discovery of novel pathway participants.
Cvijic
Mary Ellen
ME
Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA. maryellen.cvijic@bms.com
Xiao
Gutian
G
Sun
Shao-Cong
SC
D000585:Aminacrine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D001681:Biological Assay / Q000379:methods*; D003001:Cloning, Molecular; D005434:Flow Cytometry; D015723:Gene Library; D006801:Humans; D019169:Jurkat Cells; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology*; D009154:Mutation; D016328:NF-kappa B / Q000502:physiology; D009588:Nitrogen Mustard Compounds / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000187:drug effects / Q000502:physiology*; D014162:Transfection
D009153:Mutagens; D016328:NF-kappa B; D009588:Nitrogen Mustard Compounds; D000585:Aminacrine; C004294:acridine half-mustard