Journal
JOURNAL OF IMMUNOLOGICAL METHODS
Volume 278, Issue 1-2, Pages 293-304Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/S0022-1759(03)00191-1
Keywords
Jurkat T cells; somatic mutagenesis; complementation cloning; retroviral gene delivery; cDNA libraries; soft agar cloning
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Funding
- NCI NIH HHS [1F32CA83280, 2R01 CA68471] Funding Source: Medline
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Somatic cell mutagenesis is a powerful genetic approach used in dissection of signal transduction pathways ill 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 ill 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. (C) 2003 Elsevier B.V. All rights reserved.
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