Dissecting NF-κB signaling induced by genotoxic agents via genetic complementation of NEMO-deficient 1.3E2 cells

Methods in Molecular Biology
Shawn S Jackson, Shigeki Miyamoto

Abstract

The transcription factor NF-κB regulates expression of a diverse set of genes to modulate multiple biological and pathological processes. Among these, NF-κB activation in response to genotoxic agents has received considerable attention due to its role in regulating cancer cell resistance to chemo- and radiation therapy. Furthermore, induction of this pathway by endogenous damage is further implicated in normal developmental processes, such as B cell development, and premature aging, among others. This pathway also serves as a signaling model in which nuclear initiated signals (DNA damage) are communicated to a cytoplasmic target (IκB kinase and NF-κB). Several of the critical molecular events of this nuclear to cytoplasmic NF-κB signaling cascade were discovered, in part, by genetic complementation analyses of the NEMO-deficient 1.3E2 mouse pre-B cell line. This chapter describes methods used to generate and analyze such reconstitution cell systems and certain caveats that are critical for proper interpretation of NEMO mutant defects.

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