Genetic Analysis of NF-κB-Dependent Signaling Pathways in Mammalian Cells

Abstract

Reverse genetics is used to study the function of a known gene product by knockout or replacement. In contrast, forward genetics is used to identify an unknown gene, based on its function. In using forward genetics, one can knockout or gain gene expression by random mutagenesis or by introducing expression libraries, followed by selection based on function (Stark & Gudkov, 1999). As a result, a collection of mutant cell lines and cDNA reagents will be generated, which can be used to define detailed mechanisms for specific components in a particular signaling pathway. The application of forward genetics to mammalian cells has led to significant advances in understanding signaling in response to interferons (Darnell et al., 1994) and has led to an appreciation of the role of Janus kinases (JAKs) in many signaling pathways (Velazquez et al., 1992; Stark et al., 1998). The forward genetic approach has also been successfully applied to study the TGFβ receptor (Hocevar & Howe, 1996) and T-cell receptor-mediated signaling (Goldsmith & Weiss, 1987; Serafini et al., 1995; Williams et al., 1998). While biochemical studies and reverse genetics have tremendously advanced our understanding of the NF-κB-dependent signaling pathways, the ways in which a forward genetic approach has also been used to study these pathways will be discussed in this chapter.