Analysis of innate immune signal transduction with autocatalytic expression vectors.
Signalling pathways modulated by the family of IL-1/TLR receptors are central to innate immune responses. Novel components playing key regulatory roles in these pathways continue to be isolated. Here we describe the use of autocatalytic vectors for identifying critical components of these signalling pathways. The method was tested with a vector system where cDNA clones are expressed as EGFP fusion proteins, or in an IRES containing mRNA, combined with a transcription reporter. These constructs are placed under the control of an inducible promoter, responsive to activation of TIR receptors such as IL-1RI or TLR-4. cDNAs which activate the promoter will, when transcribed, form a positive feedback loop. We introduced TIR signalling pathway components into both types of vectors. The components tested regulated reporter (EGFP/luciferase) expression in both cases. Our data suggest that this type of system is capable of selective identification of components from signal transduction pathways once the promoter of a relevant inducible gene is identified from, for example, micro-array experiments.
D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007113:Immunity, Innate / Q000235:genetics; D053583:Interleukin-1beta / Q000378:metabolism*; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D017472:Receptors, Interleukin-1 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D013997:Time Factors; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D053583:Interleukin-1beta; D016209:Interleukin-8; D012333:RNA, Messenger; D017472:Receptors, Interleukin-1; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases