An inducible system for analysis of Topo IIα mutants. (A, left) Schematic describing the strategy to derive HeLa EM2-11ht cells expressing RNAi-resistant mCherry–Topo IIα at close to endogenous levels. Single copy insertion of Topo IIα alleles at locus 5q31.3 was achieved using asymmetric FLP (F/F3) recombinase sites (see Materials and methods and Weidenfeld et al., 2009). (A, right) Immunoblot detecting endogenous Topo IIα and exogenously expressed mCherry–Topo IIα. HeLa EM2-11 cells were infected with lentiviral particles encoding knockdown shRNA constructs at day 0 against Topo IIα and Topo IIβ simultaneously with the addition of 250 ng/ml doxycycline to induce expression of the exogenous allele. Endogenous Topo IIα is effectively depleted, whereas the exogenous protein is expressed to approximately endogenous levels within 24 h. (B) Localization of mCherry–Topo IIα and mCherry–Topo IIα ΔChT after 48 h of induction with doxycycline in live HeLa EM2-11 cells (top) or after fixation with methanol (bottom). Topo IIα ΔChT is labile upon fixation. Bars, 10 µm.