ZFN-facilitated gene targeting at the ADH1 locus. (A) A schematic of the ADH1 locus is shown with exons depicted as black boxes. The donor plasmid shown below the locus contains a left homology arm (LHA) of 161 bp and a right homology arm (RHA) of 144 bp. Successful HR will result in an insertion of 68 bp and deletion of 12 bp at the ZFN cut site. (B) PCR-based detection of HR. Protoplasts derived from the five genotypes were treated with donor only, donor and estradiol, and estradiol only. As depicted in panel A, the ZY070-F and ZY073-R1 (F + R1) primer set was used to detect HR events. Amplification of the locus with the ZY070-F and ZY070-R (F + R) primer set was used as a PCR control. (Arrows) Amplification products due to HR. This experiment was repeated twice with similar results.