Figure 3.

Effect of repetitive DNA sequences in a direct or inverted repeat configuration on homology-directed gene repair in HeLa cells. (A) HeLa cells were transfected with acceptor^DR.1 or acceptor^IR.1 alone (DR.1 and IR.1, respectively) or with either of these two acceptor plasmids in combination with the donor construct GFP^ΔATG (donor + DR.1 and donor + IR.1, respectively). Mock-transfected HeLa cells (mock) and HeLa cells transfected with GFP^ΔATG alone (donor) or together with acceptor^ScR (donor + ScR) served as negative controls. The positive control for the rescue of GFP expression by HR was provided by cotransfecting HeLa cells with acceptor^ScR, GFP^ΔATG and the I-SceI-encoding plasmid pCAG.I-SceI (donor + ScR + I-SceI). Quantification of the number of GFP-positive cells was carried out by flow cytometry at 4 days post-transfection. A minimum of 5 and a maximum of 11 independent experiments were performed with 10 000 events corresponding to viable cells being measured per sample. (B) HeLa cells were cotransfected with GFP^ΔATG plus either acceptor^IR.2, acceptor^DR.2, acceptor^IR.3, acceptor^DR.3, acceptor^IR.4 or acceptor^DR.4. To facilitate comparison, data sets corresponding to HeLa cells cotransfected with GFP^ΔATG and acceptor^ScR or with GFP^ΔATG, acceptor^ScR and pCAG.I-SceI as well as those corresponding to HeLa cells cotransfected with GFP^ΔATG and either acceptor^IR.1 or acceptor^DR.1 presented in Figure 3A are repeated in Figure 3B (open and gray bars, respectively). Quantification of GFP expression rescue was carried out by flow cytometry at 4 days post-transfection. Cumulative data from 4 different experiments (solid bars) are expressed as mean ± standard deviation. *P = 0.002, **P = 0.009. (C) Flow cytometric analysis of HeLa cells that, in addition to being exposed to the donor plasmid GFP^ΔATG also, received acceptor^ScR, acceptor^IR.1 or acceptor^spIR.1. In the latter construct, the inverted repeat of test sequence 1 is interrupted at its axis of symmetry by an I-SceI recognition site (see diagram below the graph). HeLa cells cotransfected with GFP^ΔATG, the I-SceI encoding plasmid pCAG.I-SceI and either acceptor^ScR or acceptor^spIR.1 served as positive controls for HR-mediated GFP repair. Data corresponding to a minimum of three different experiments are shown as mean ± standard deviation.