The CCR5 locus in macaque HSPCs is efficiently targeted by macaque-specific CCR5 (mCCR5) ZFNs ex vivo. (A) Schematic of the CCR5 locus showing TM, CL, and EL domains. Single, double, and triple bars indicate sites of a previously published human CCR5 ZFN target region, macaque CCR5 ZFN target region, and the human Δ32 mutation, respectively. The lower schematic shows the macaque CCR5 coding sequence, highlighting the target sites for the ZFN pair 32106 (ZFN-L) and 32118 (ZFN-R). (B) At the indicated times after electroporation of macaque CCR5 ZFN mRNA into macaque HSPCs, cells were collected and western blots were performed for ZFN and actin protein. (C) Representative Cel I nuclease assay demonstrating robust targeting of mCCR5 in HSPCs following delivery of mCCR5 ZFN mRNA. The percentage of disrupted mCCR5 alleles, as measured by densitometry, is indicated below each lane. CL, cytoplasmic loop; EL, extracellular loop; GFP, green fluorescent protein; Mock, control cells that were not electroporated; TM, transmembrane.