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

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

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Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada.


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.

[Indexed for MEDLINE]

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