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PubMed · 16503394

Functional screening for proapoptotic genes by reverse transfection cell array technology.

Otto Mannherz·Daniel Mertens·Meinhard Hahn·Peter Lichter
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Abstract

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.

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ApoptosisApoptosis Regulatory ProteinsCell LineFlow CytometryGene Expression ProfilingGenesHumansIn Situ Nick-End LabelingOpen Reading FramesPhenotypeTissue Array AnalysisTransfectionJournal ArticleResearch Support, Non-U.S. Gov'tValidation Study

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Otto Mannherz, Daniel Mertens, Meinhard Hahn, Peter Lichter. 2006-02-28. Functional screening for proapoptotic genes by reverse transfection cell array technology.. https://doi.org/10.1016/j.ygeno.2005.12.009

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