Figure 3.

Single-cell model of MtnA gene activation. (A) Schematic for the generation of transgenic cell lines. The reporter plasmid is transfected into S2 cells together with a small amount of hygromycin-resistant marker plasmid. After the generation of stable cell lines, fluorescence in situ hybridization (FISH) was conducted to confirm the integration of reporter plasmids. (B) Luciferase assay conducted to confirm that transcription from the transgenes exhibits low basal activity but is highly inducible upon induction by copper. (C) Primer extension assay was conducted using 20 μg of total RNA from uninduced (−) and induced (+) transgenic cells. A single major transcription start site was observed upon induction. (D) These panels show FISH experiments testing the size and response of the MtnA transgenic cluster. The top row shows images of MTF-1 (green on the left, red on the right) and DNA staining (DAPI). The middle panels mark the transgenic cluster with DNA FISH (red) on the left and RNA FISH (green) on the right. The bottom row shows the merge of the two images. MTF-1 is recruited to the transgenic cluster upon induction with copper (bottom right), and the cluster is active only upon induction (bottom left).