Figure 5.

In situ cleavage of genomic target site by transferrin-ZFN1/transferrin-ZFN2 pair. (A) Schematic diagram of the U2OS 2-6-3 transgene array, modified from (20). Approximately 200 copies of this array are integrated at a single genomic site. Each repeat in the tandem array contains 256 copies of the lac operator recognition site and a single ZFN-cleavable CFP cDNA sequence. The transgene is not induced in these experiments, and several other elements not used here, and relevant only to gene expression are depicted in gray: 96 copies of a tetracycline response element, a minimal CMV promoter, a peroxisomal targeting signal (SKL), 24 copies of the MS2 translational operator, a rabbit beta-globin intron/exon module and a polyadenylation signal (20). (B) Merged images of fluorescent lac repressor (LacI-ECFP) and the DSB marker 53BP1 in untreated control cells (top) or in two representative fields of transferrin-ZFN treated cells (two lower rows). Insets show colocalization of Laci-ECFP and anti-53BP1 staining in treated cells. (C) Tabulation of co-localization in the indicated numbers of untreated and transferrin-ZFN treated U2OS 2-6-3 cells.