Integrated measurement of split TEV and cis-regulatory assays using EXT encoded reporter libraries

Methods Mol Biol. 2012;812:309-23. doi: 10.1007/978-1-61779-455-1_19.

Abstract

Intracellular signaling initiated by extracellular ligands that activate cell surface receptors is a complicated process that involves multiple interconnected biochemical steps. Protein-protein interactions are often regulated by activated kinases via phosphorylation of specific residues. Such transient regulated interactions are central to many signaling cascades. Downstream signaling converges at the level of transcription factors to finally regulate adaptive transcriptional responses. There are powerful methods available to study transcriptional changes even at a global level, however, measuring upstream regulatory mechanisms is still challenging. We designed an experimental approach termed EXTassay that enables the parallel analysis of signaling events upstream of gene expression. We make use of different types of reporter gene assays that are invariably linked to unique expressed oligonucleotide tags (EXTs) serving as quantitative decoders of respective assays. EXT-reporters can be introduced into living cells and analyzed in pools by microarray hybridization or sequencing.

MeSH terms

  • Animals
  • Base Sequence
  • Cloning, Molecular
  • DNA, Complementary / biosynthesis
  • DNA, Complementary / genetics
  • Expressed Sequence Tags / metabolism*
  • Gene Library*
  • Genes, Reporter / genetics*
  • PC12 Cells
  • Protein Interaction Mapping / methods*
  • Rats
  • Regulatory Sequences, Nucleic Acid / genetics*
  • Transfection

Substances

  • DNA, Complementary