Functional screening for proapoptotic genes by reverse transfection cell array technology

Genomics. 2006 May;87(5):665-72. doi: 10.1016/j.ygeno.2005.12.009. Epub 2006 Feb 28.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Apoptosis / genetics
  • Apoptosis / physiology
  • Apoptosis Regulatory Proteins / genetics*
  • Cell Line
  • Flow Cytometry
  • Gene Expression Profiling
  • Genes
  • Humans
  • In Situ Nick-End Labeling
  • Open Reading Frames
  • Phenotype
  • Tissue Array Analysis / methods*
  • Transfection / methods*

Substances

  • Apoptosis Regulatory Proteins