Zinc-finger nuclease-assisted targeting of the Rosa26 locus in ES cells. (A) Targeting vector pRosa26.11 for insertion of a hygromycin/puromycin resistance cassette, into the Rosa26 locus. The structure of the wild-type locus, including the ZFN^Rosa recognition sites that overlap with an intronic XbaI site (X), and of the recombined Rosa26 allele are shown. The location of the Rosa26 promoter (Pr.), first exon, of the 5′-Rosa Southern blot probe and of EcoRV (E) sites and fragments are indicated. (B) Southern blot analysis of EcoRV digested genomic DNA of 32 hygromycin resistant ES cell clones transfected with pRosa26.11 using the Rosa26 5′ probe. The wild-type Rosa26 locus exhibits a 11.5-kb EcoRV fragment (WT). (C) Southern blot analysis of EcoRV digested genomic DNA of 34 hygromycin resistant ES cell clones transfected with pRosa26.11 and ZFN^Rosa expression vectors using the Rosa26 5′ probe. Targeted integration of the resistance gene cassette is indicated by the presence of an additional 4.5-kb EcoRV fragment.

Targeted integration of a Venus reporter gene into the Rosa26 locus. (A) Targeting vector pRosa26.3 for insertion of a 1.1-kb Venus gene, including a splice acceptor (SA) and polyA site, into the Rosa26 locus. The structure of the wild-type locus, including the ZFN^Rosa recognition sites that overlap with an intronic XbaI site (X), and of the recombined Rosa26 allele are shown. The location of the Rosa26 promoter (Pr.), first exon, of Southern blot probes and XbaI (X) and BamHI (B) sites and fragments are indicated. (B) Genomic DNA of E18 fetuses derived from zygotes coinjections of ZFN^Rosa and pRosa26.3 was digested with BamHI or XbaI and analyzed by Southern blotting using the Rosa26 5′ probe. The wild-type Rosa26 locus exhibits a 5.8-kb BamHI and a 4.7-kb XbaI band. Targeted integration of the reporter gene is indicated by the presence of a predicted 3.1-kb BamHI and a 5.6-kb XbaI fragment detected with the Rosa26 5′ probe (fetus #6). The loss of the ZFN^Rosa target XbaI site by NHEJ in nonrecombined alleles results in a 9.0-kb band (fetus #6). (C) Analysis of BamHI digested DNA with the internal Venus probe shows a predicted 3.9-kb band from the recombined allele of fetus 6. (D) Habitus of fetus #6 (recombined) and #7 (wild type).

Principle of ZFN-assisted gene targeting in zygotes. Zygotes collected from wild-type mice are coinjected into the pronucleus and cytoplasm with DNA of a gene targeting vector and mRNAs for the expression of a pair of gene specific zinc-finger nucleases (ZFN1/2). HR of the targeting vector with the target site results in a knockout (KO) or knock-in (KI) allele. Manipulated zygotes are subsequently transferred into pseudopregnant females to recover mutant mice.

Targeted Integration of a β-Galactosidase reporter gene into the Rosa26 locus. (A) Targeting vector pRosa26.8 for insertion of a 4.2-kb β-Galactosidase gene, including a splice acceptor (SA) and polyA site, into the Rosa26 locus. The structure of the wild-type locus, including the ZFN^Rosa recognition sites that overlap with an intronic XbaI site (X), and of the recombined Rosa26 allele are shown. The location of the Rosa26 promoter (Pr.), first exon, of Southern blot probes and EcoRV (E), XbaI (X), and SwaI (S) sites and fragments are indicated. (B) Genomic DNA of E18 fetuses derived from zygote coinjections of ZFN^Rosa and pRosa26.8 was digested with XbaI or EcoRV and analyzed by Southern blotting using the Rosa26 5′ probe. The wild-type Rosa26 locus exhibits a 4.7-kb XbaI and a 11.5-kb EcoRV fragment (WT). The loss of the ZFN^Rosa target XbaI site by NHEJ results in a 9.0-kb fragment (fetus #14, #15, and #21). Targeted integration of the reporter gene is detected with the Rosa26 5′ probe by the presence of a 8.5-kb XbaI and 3.6-kb EcoRV fragment (fetus 22). (C) Sequence comparison of cloned PCR products (primers Rosa 5HA/Rosa 3HA), covering the ZFN^Rosa target region, from genomic DNA of fetus #9, #14, and #21, with the respective Rosa26 wild-type sequence. The location of the XbaI site within the ZFN^Rosa target region is indicated. In PCR amplified Rosa26 alleles from pups #9, #14, and #21, the XbaI site is lost by the deletion of multiple nucleotides (dashes). Sequence identity of all compared sequences is marked in yellow and partial sequence marked in blue; deleted nucleotides, as compared to the Rosa26 wild-type sequence, are indicated by a dash (-). Fetus #9 exhibited by Southern blot analysis an almost homozygous loss of the ZFN target XbaI site (Fig. S1A). (D) Southern blot analysis of EcoRV + SwaI digested genomic DNA with the Rosa26 3′ probe reveals a 11.5-kb band derived from the wild-type locus and a 9.4-kb fragment from the recombined allele (fetus #22 and #14). Exposure times for samples 21–55 were 2 days (2 d) and 3 days (3 d) for sample 14. Southern blot analysis of XbaI digested DNA of fetus #22 with an internal β-Galactosidase probe shows the predicted 8.5-kb band representing the recombined allele. (E) Habitus of fetus 22 (recombined) and 24 (wild type) and X-Gal staining of liver sections.