title: Development of a gene-trap vector with a highly sensitive fluorescent protein reporter system for expression profiling.
abstract: 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.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada. ttanaka@uiuc.edu
authors.forename: Tetsuya S
authors.initials: TS
authors.lastname: Tanaka
authors.forename: Ryan E
authors.initials: RE
authors.lastname: Davey
authors.forename: Qing
authors.initials: Q
authors.lastname: Lan
authors.forename: Peter W
authors.initials: PW
authors.lastname: Zandstra
authors.forename: William L
authors.initials: WL
authors.lastname: Stanford
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D015152:Blotting, Northern; D017931:DNA Primers / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017403:In Situ Hybridization; D008164:Luminescent Proteins / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; C425301:NKX6-2 protein, human; C485184:enhanced green fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins