Functional screening for proapoptotic genes by reverse transfection cell array technology

Application of mathematical algorithms to sequenced whole genomes revealed a large number of predicted genes, requiring functional assays for their characterization in a high-throughput manner. Here, we report on the development of a screening assay, which is based on reverse transfection of cellular arrays and subsequent analysis of cell morphology to identify novel proapoptotic genes. Expression plasmids containing full-length cDNAs were cotransfected with the reporter plasmid pEYFP to screen for apoptotic body formation, based on EYFP fluorescence. The assay was validated and applied to 382 human sequence-verified full-length open reading frames, most of them of unknown function. In this initial screening, proapoptotic effects could be demonstrated for 10 of these genes. For 6 of them apoptosis induction could be confirmed both by TUNEL assay and by FACS analysis of cells stained according to Nicoletti: 1 gene was not yet annotated for an apoptotic function (ST6GAL2), while 5 genes were without annotated function (FLJ20551, CXorf12, FAM105A, TMEM66, C19orf4). Our study demonstrates the potential of this method to characterize functionally genes of unknown function in a highly parallel format.