Figure 1. KRAB–mediated silencing can act over several tens of kilobases.

(A) Mechanism of how endogenous genes are targeted by tTRKRAB using the lentiviral vector-based “Trapping/Silencing” (TrapSil) system: TetO-containing gene traps carrying the promoterless puro^R-GFP gene only express this reporter if after proviral integration they “trap” an actively transcribing gene. The TetO sites further allow binding of the ectopic repressor tTRKRAB to the gene traps after dox removal, while the trap reporter serves as a direct read-out for the effects of tTRKRAB–mediated “silencing”. (B) tTRKRAB–expressing HeLa cells were transduced with LV TrapSil vectors and 23 clones expressing the trap reporter were isolated. Mean fluorescence intensity (MFI) GFP values of these individual clones, cultured with and without dox, were determined and the ratios of these values were used to calculate the silencing efficiency (% silencing  = 1- ((MFI GFP –dox)/(MFI GFP +dox))) depicted below the x-axis for each clone. (C) GFP-mediated cell sorting was used to isolate populations of HeLa cells exhibiting either a “repressible” (>90% silencing) or an “irrepressible” (<10% silencing) phenotype. 484 repressible and 699 irrepressible clonal integrations were mapped and the distance between them and their trapped promoter was plotted on a cumulative histogram.