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 r andom library sequencing projects is leading to the rapid identification of many putative genes, virtual transcripts and ESTs of unknown function. The re is therefore an increasing need for high throughput, sensitive and robus t methods for identification and characterisation of genes, and/or their pr oducts, based on function. We describe a high throughput functional express ion screen based on semi-quantitative analysis of enhanced green fluorescen t 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 plasmi ds gave a detectable signal above background at a pool size of 250-500. Rep licate tests indicate that the assay is reproducible at this pool size. At this level we demonstrate that large (>10(6) transformants) libraries can b e feasibly screened. (C) 2000 Elsevier Science B.V. All rights reserved.