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.
Mannherz
Otto
O
Division of Molecular Genetics (B060), Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
Mertens
Daniel
D
Hahn
Meinhard
M
Lichter
Peter
P
D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D002460:Cell Line; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005796:Genes; D006801:Humans; D020287:In Situ Nick-End Labeling; D016366:Open Reading Frames; D010641:Phenotype; D046888:Tissue Array Analysis / Q000379:methods*; D014162:Transfection / Q000379:methods*
D051017:Apoptosis Regulatory Proteins