| First Author | Tanaka TS | Year | 2008 |
| Journal | Genesis | Volume | 46 |
| Issue | 7 | Pages | 347-56 |
| PubMed ID | 18615730 | Mgi Jnum | J:138547 |
| Mgi Id | MGI:3805394 | Doi | 10.1002/dvg.20404 |
| Citation | Tanaka TS, et al. (2008) Development of a gene-trap vector with a highly sensitive fluorescent protein reporter system for expression profiling. Genesis 46(7):347-56 |
| abstractText | Summary: 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. |
