Fig. 1. Visualization of the nuclear sites of ss to ds AAVLacO.14 conversion (rAAV foci) in live GFP-LacR cells. (A) AAVLacO.14 contains the AAV ITRs flanking 14 copies of a sequence corresponding to an 8-mer of the Lac operator sequence cassette (LacO), for a total of 112 LacR binding sites. Inside the transduced cells, the GFP-LacR protein only binds the vector genome after its conversion to dsDNA. (B) Selected images from a time-lapse series of an individual MRC/GFP-LacR cell treated with HU and observed from 3 to 28 hours post infection (p.i.). The location of rAAV foci was monitored through a z-series of images. Along the z axis, images of nuclei were captured at 0.25 µm intervals, and the final images were obtained by projection of the individual sections. A movie showing the formation of these foci in real time is presented in the supplementary material Movie 1. (C) Formation of rAAV foci in MRC/GFP-LacR cells transduced with AAVLacO.14 but not with AAV-LacZ. Representative images taken 24 hours after transduction with the same amounts of vector are shown. Cells were either untreated (nt) or treated with hydroxyurea (HU), camptothecin (campto) or bleomycin (bleo), in conditions known to increase the efficiency of rAAV transduction (Zentilin et al., 2001) (see also Materials and Methods).