Multiple termination sites enhance transcription <i>in vivo</i>.

<p>(A) Reporter plasmids containing the rDNA promoter, <i>Firefly</i> luciferase and either no (pTΔ), one (pT₁), two (pT₂), ten (pT₁₀) termination sites and T₁ and T_1–10 in reverse orientation (pT_1r and pT_10r) were co-transfected with a <i>Renilla</i> luciferase encoding plasmid (pRL-TK) into CHO cells. As a control, empty pBluescript vector was co-transfected. Transcriptional activities were analysed using a dual luciferase reporter assay. The ratio <i>of Firefly</i>/<i>Renilla</i> relative light units (RLU) of three independent experiments is given. Error bars indicate standard deviations. The functional elements and the sizes of the reporter plasmids are depicted. (B) Reporter plasmids were co-transfected with a GFP-TTFΔN348 expression vector and analysed as described in (A). (C) Reporter plasmids were co-transfected with a GFP-TTFΔN470 expression vector and analysed as described in (A). (D) RNA FISH using CHO cell lines with stably integrated rDNA minigenes. CHO-pT₁₀ cells containing an rDNA minigene with a full terminator, were stained with DAPI (in blue in the middle panel), with α-B23 antibody staining the nucleoli (left panel; shown in red in the middle panel), and integrated reporter gene transcripts were visualized by FISH (right panel; shown in green in the middle panel). Bar: 5 µm. (E) Transcription levels of genomically inserted pT₁ and pT₁₀ constructs were assayed using RT-qPCR. Comparative quantitation was performed and RNA levels of the <i>Firefly</i> luciferase sequence were normalized to β-actin expression. Relative transcript levels of three independent experiments are given in relation to non-transfected CHO Flp-In cells (control), error bars denote standard deviations.