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Abstract: In this study, cell microarray technology is used to identify novel human genes associated with CRE pathway activation. By reverse transfection, expression plasmids containing full-length cDNAs were cotransfected with the reporter plasmid pCRE-d2EGFP to monitor the activation of the CRE pathway via enhanced green fluorescence protein (EGFP) expression. Of the 575 predominantly novel genes screened, 22 exhibited relatively higher EGFP fluorescence compared with a negative control. After a functional validation with a dual luciferase reporter system that included both cis- and trans-luciferase assays, 4 of the 22 genes (RNF41, C8orf32, C6orf208, and MEIS3P1) were confirmed as CRE-pathway activators. Western blot analysis revealed that RNF41 can promote CREB phosphorylation. These results demonstrate the successful combination of cell microarray technology with this reporting system and the potential of this tool to characterize functions of novel genes in a highly parallel format. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]