A novel mammalian expression screen exploiting green fluorescent protein-based transcription detection in single cells.
The accumulation of DNA sequence information from large-scale genomic and random library sequencing projects is leading to the rapid identification of many putative genes, virtual transcripts and ESTs of unknown function. There is therefore an increasing need for high throughput, sensitive and robust methods for identification and characterisation of genes, and/or their products, based on function. We describe a high throughput functional expression screen based on semi-quantitative analysis of enhanced green fluorescent protein expression in single cells by confocal microscopy. The assay was implemented in a micro-scale format, requiring around 10(4) cells/test. The system was validated by co-transfection of a series of cDNAs encoding pro-inflammatory cytokine intracellular signal mediators with a d2EGFP reporter containing a cytokine responsive promoter. The majority of the test plasmids gave a detectable signal above background at a pool size of 250-500. Replicate tests indicate that the assay is reproducible at this pool size. At this level we demonstrate that large (>10(6) transformants) libraries can be feasibly screened.
Kiss-Toth
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E
Functional Genomics Group, Division of Molecular & Genetic Medicine, University of Sheffield, Royal Hallamshire Hospital, M Floor, S10 2JF, Sheffield, UK.
Guesdon
F M
FM
Wyllie
D H
DH
Qwarnstrom
E E
EE
Dower
S K
SK
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics; D029681:Arabidopsis Proteins; D044943:Fatty Acid Desaturases / Q000235:genetics; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007375:Interleukin-1 / Q000276:immunology; D053592:Interleukin-1 Receptor-Associated Kinases; D016209:Interleukin-8 / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics*; D008322:Mammals; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011506:Proteins / Q000235:genetics; D011971:Receptors, Immunologic; D017472:Receptors, Interleukin-1 / Q000235:genetics; D015398:Signal Transduction / Q000276:immunology; D048029:TNF Receptor-Associated Factor 6; D051996:Transcription Factor RelA; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology; D000097782:NF-kappaB-Inducing Kinase
D048868:Adaptor Proteins, Signal Transducing; D000943:Antigens, Differentiation; D029681:Arabidopsis Proteins; D044943:Fatty Acid Desaturases; D049452:Green Fluorescent Proteins; D007375:Interleukin-1; D053592:Interleukin-1 Receptor-Associated Kinases; D016209:Interleukin-8; D008164:Luminescent Proteins; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D011971:Receptors, Immunologic; D017472:Receptors, Interleukin-1; D048029:TNF Receptor-Associated Factor 6; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000097782:NF-kappaB-Inducing Kinase; C507779:MYD88 protein, human; C084083:Fad7 protein, Arabidopsis