Figure 1.

General scheme for forward genetics analysis of NF-κB-dependent signaling pathway, using lethal selection to identify regulative proteins. 293-TK/Zeo cells, carrying both TK and Zeo selectable markers, can be used in conjunction with chemical mutagenesis (left) or insertional mutagenesis (right) to obtain mutants unresponsive to a stimulus or constitutive mutants, and then to identify either stimulus-specific signaling components or general activators or repressors. A cDNA library (middle) can also be overexpressed to screen directly for activators or repressors. Additionally, constitutive mutants can be further mutated by insertional mutagenesis using VBIM (bottom) to screen for mutants with low NF-κB to identify activators or repressors.