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. Author manuscript; available in PMC: 2013 Apr 5.
Published in final edited form as: Nat Methods. 2011 Jul 17;8(9):753–755. doi: 10.1038/nmeth.1653

Figure 2.

Figure 2

Deletion of chromosomal segments using ssODNs and ZFNs at the human AAVS1 locus. (a) General ssODN design rules for deletion of chromosomal segments relative to the ZFN cut site. Sequence distal to the ZFN cleavage site (purple) and DNA sequence containing the ZFN half-site farthest from the distal deletion sequence (green) are shown. (b) ssODN sequence used to delete 5 kb upstream of the AAVS1 ZFN cut site. The ZFN binding half-site is underlined. (c) Agarose gel separation of amplified genomic DNA from K562 cells transfected with the following constructs 2 d after nucleofection (1, ssODN plus construct encoding ZFN; 2, ssODN only; and 3, construct encoding ZFN only; see supplementary note 2 for ssODN sequence). The expected fragment sizes of the wild-type and deletion alleles are indicated. PCR fragments greater than 1.5 kb were not detected under the experimental conditions. M, DNA marker (Sigma); *3′ deletion from the ZFN cut site; **5′ and 3′ deletion off the ZFN cut site by transfecting both d5-AAVS1-0.1kb and d3-AAVS1-0.1kb deletion ssODNs; supplementary note 2); all other lanes are 5′ deletions from the cut site.