Somatic cell genetics for the study of NF-kappaB signaling in innate immunity.
Vertebrates have evolved acquired immunity, but to detect an infection in its early stages they, nonetheless, rely on Toll-like receptors (TLRs) and other innate immune receptors. We have performed genomewide mutagenesis screens in an immortalized murine cell line to study nuclear factor kappaBeta (NF-kappaB) signaling in the context of innate immunity. To enable metabolic and physical selection for alterations in NF-kappaB signaling, we equipped cells with multiple reporter genes. Despite the diploid nature of the cells, multiple mutants unresponsive to lipopolysaccharide and CpG DNA were isolated from as few as 10 million mutagenized cells. Mutant clones may lead to the discovery of novel genes, and in combination with syngeneic wild-type reporter cells, they may allow a detailed functional analysis of NF-kappaB signaling. Compared with the use of whole animals in genetic screens, somatic cell genetics allows the isolation of genes required for innate immunity, even if these genes also have an essential function in development. Our discovery of an essential role for the endoplasmic reticulum chaperone gp96 (Grp94) in the maturation of TLRs and our work on the regulation of the inhibitor of nuclear factor kappaB kinase (IKK) complex by Nemo will be discussed in this context.
Krumbach
Rebekka
R
Medical Research Council Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Hills Road, Cambridge CB2 2QH, UK.
Bloor
Stuart
S
Ryzhakov
Grigory
G
Randow
Felix
F
D000818:Animals; D015226:Dinucleoside Phosphates; D017930:Genes, Reporter; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism; D007113:Immunity, Innate; D008562:Membrane Glycoproteins / Q000502:physiology; D009154:Mutation; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D015398:Signal Transduction; D051193:Toll-Like Receptors / Q000276:immunology / Q000502:physiology*; D054875:Ubiquitination
D015226:Dinucleoside Phosphates; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D051193:Toll-Like Receptors; C053387:endoplasmin; C018309:guanylyl-(3'-5')-guanosine; D051550:I-kappa B Kinase