Development of a gene-trap vector with a highly sensitive fluorescent protein reporter system for expression profiling

Genesis. 2008 Jul;46(7):347-56. doi: 10.1002/dvg.20404.


Combining high-content screening (HCS) with random gene-trap mutagenesis could be a powerful tool to investigate transcriptional networks, cell signaling, chemical genetics, and developmental processes. However, a critical limitation has been poor quantification of reporter expression per cell. To overcome this hurdle, we generated a variety of Gtx-based expression cassettes and re-evaluated translational enhancement of arrayed Gtx segments in tandem by HCS. We then modified the cassette into a new polyA trap vector, which consists of a variant of yellow fluorescent protein, Venus, in combination with the Gtx segments. Expression of Venus was detected in about 60% of trapped genes assayed in embryonic stem cell (ESC) cultures, comparable to expression screening of LacZ-based vectors. Furthermore, tetraploid aggregations using a clone encoding a gene-trap insertion into Twist2 demonstrated identical spatiotemporal expression between Venus and Twist2. This highly sensitive reporter system is amenable to high-throughput expression-based real-time HCS including single cell analyses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / metabolism*
  • Blotting, Northern
  • DNA Primers / genetics
  • Embryonic Stem Cells / metabolism*
  • Gene Expression Profiling / methods*
  • Genetic Vectors / genetics*
  • Green Fluorescent Proteins / metabolism
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism*
  • Humans
  • In Situ Hybridization
  • Luminescent Proteins / metabolism*
  • Mutagenesis / genetics


  • Bacterial Proteins
  • DNA Primers
  • Homeodomain Proteins
  • Luminescent Proteins
  • NKX6-2 protein, human
  • enhanced green fluorescent protein
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins