Transcription of the targeted locus at the nuclear lamina. (A and B) Representative images used for counting cells to quantify the percentage of cells showing transcription in control or targeting conditions. Control cells or targeting cells were transiently transfected with pVitro2–Tet-on + MS2-YFP (green), induced for 12 h with cumate for targeting fusion expression (red, arrows), and then incubated in the absence (a–c) or presence (d–f) of Dox for 5 h. In the Dox (+) condition, MS2-YFP accumulated at the site of the targeted locus (B, e, green enrichment, arrow) and MS2-YFP localized with the locus (B, f, arrow). In the presence of Dox, ∼90% of control cells and ∼70% of targeting cells were transcriptionally active. Arrows in A (b) and B (b) represent absence of accumulation of MS2-YFP. n = 100 nuclei per experiment for each control and test. Bars, 5 μm. (C) Still images (a–p) from time series (0–5 h) of targeting cells showing transcription at the lamina. At 0 h, in Dox (−) condition, targeted locus (a and d, arrows) showed no accumulation of MS2-YFP (b and d, arrows) and no CFP-SKL protein in peroxisomes (c and d). In Dox (+) condition, transcription was observed at the targeted nuclear lamina as observed by the accumulation of MS2-YFP (f, j, and n, arrows) and CFP-SKL localization (k, l, o, and p) over time. Images are projections of deconvolved z stacks taken every 20 min. Arrows: (e, i, and m) decondensed targeted locus; (h, l, and p) accumulation of MS2-YFP. Bar, 5 μm. See Video 4 for the entire time series.