a, F9 ZFNs target intron 1 of the human F9 gene, allowing for homology-directed repair upstream of 95% of F9 mutations. b, ZFN expression constructs (400 ng) were transfected (right lane) or not (left lane) into K562 cells and genomic DNA was harvested 3 days post-transfection. The Cel-I assay was used to determine the frequency of ZFN-induced insertions and deletions (indels) in both samples, indicated as the % Indels at the base of the right lane. ZFN (FLAG-tagged) expression is confirmed by α-FLAG immunoblotting, and αNFκB-p65 serves as a loading control. c, Schematic of RFLP assay detailing ZFN-mediated targeting of a NheI restriction site tag to the human F9 gene. d, Co-transfection of 400 ng of ZFN expression plasmid with increasing amounts of NheI donor plasmid (0–4 µg) results in increasing levels of homology-directed repair (HDR) at days 3 and 10 post-transfection, whereas transfection of the NheI donor alone (4 µg) does not result in detectable HDR. Black arrows denote NheI-sensitive cleavage products resulting from HDR. PCR performed using 32P-labeled nucleotides, followed by PAGE and band intensity quantification by autoradiography. Lanes with no quantification had no detectable HDR.