Figure 1. Detecting transcription in vivo using fluorescence microscopy.

(a) Schematic of the gene cassette¹⁷ stably integrated into chromosomes of human U2OS cells. P above protein sequence denotes Pol II phosphorylation state (red, phosphorylated). Reverse tet transactivator (rtTA) in the presence of doxycycline drives gene expression from a minimal CMV promoter¹⁷. Arrows indicate the 3.3-kb region transcribed by Pol II and the 2.3-kb region labeled by GFP-MS2 fusion proteins. Red lines indicate targets of FISH oligonucleotide probes. (b–m) Active transcription sites recruit Pol II. In b,e,h,k, RFP-LacI labels gene locus. Immunofluorescence (using indicated antibodies) reveals Pol II in three phosphorylation states: unphosphorylated (c), phosphorylated at Ser5 (f) and phosphorylated at Ser2 (i). l shows that the transcription site recruits YFP–Pol II (YFP-RPB1αAm^r). In n–y, nascent mRNAs were detected at active sites. In n,r,v, CFP-LacI labels gene locus. In o,s, mRNAs bound by GFP-MS2 were detected by FISH (probes at 5' and 3' ends are shown in p,t). FISH signals at exon (w) and intron regions (x) colocalize only at transcription site (see merge of each row, q,u,y). Scale bars, 5 µm.