| 研究実績の概要 |
Previous stages of the project resulted in the identification of 3 critical Transcription Factors Binding Sites (TFBS), which conferred luciferase signal upon Nthy-ori 3-1cell cotransfection with the reporter plasmid and RET/PTC1 or BRAFV600E-encoding vectors. A library of artificial promoters was prepared, which included either single-copy or concatemerized individual TFBS or a mixture of those. These promoters were cloned into pZsGreen1-DR plasmid as the goal was to establish a fluorescent protein-based cell reported system.
In order to determine how the promoter activity in the luciferase assay is translated into the short-living GFP signal, the library of 46 artificial promoter/ZsGreen1-DR reporter plasmids was screened using transient cell cotransfection with small amounts of RET/PTC1 and BRAFV600E-encoding vectors. Acceptable, in terms of signal-to-noise and specificity, green fluorescence was observed for seven artificial promoter/ZsGreen1-DR constructs.
These plasmids were stably transfected into Nthyr-ori 3-1 and clones were selected with neomycin. Only those subclones with negligible background fluorescence were further used.
Subcloned cells were challenged with RET/PTC1 and BRAFV600E oncogene-encoding vectors, and the induction of GFP signal was observed in three cell lines by live cell imaging.
The proof of principle study was reasonably successful.
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