FIG. 2.

Effect of cytostatic drugs on DNA double-strand break-stimulated gene targeting. (a) Setup of DSB-mediated gene correction. The target locus (TL) contains a disrupted EGFP (mGFP) gene with binding sites for I-SceI and zinc-finger nucleases (ZFNs). After infection with vector AAV.donor-SceI, the I-SceI–induced DSB stimulates homologous recombination with the AAV donor to restore EGFP (cTL). AAV.donor-REx expresses DsRed-Express and served as a control vector. Small arrows indicate primer binding sites for qPCR analysis (Fig. 4). (b–d) Rescue of EGFP expression in HeLa (b), HT-1080 (c), and U-2 OS cells (d). A total of 50,000 cells were treated with cytostatic drugs, as indicated, before infection with AAV.donor-SceI (2,000 gc/cell). The extent of gene correction was measured by counting EGFP-positive cells by flow cytometry four days after transduction. Graphs indicate average and standard deviation of three independent experiments. Asterisks mark significant differences (p<0.05) from nontreated cells. IRES, internal ribosome entry site; NeoR, neomycin resistance gene; wpre, woodchuck hepatitis post-transcriptional regulatory element; cTL, corrected target locus; E, etoposide; H, hydroxyurea; I, indirubin-3′-monoxime; M, L-mimosine; V, vinblastine; w/o, without cytostatics.