TALEN/ZFN-generated 5.5-Mb deletions on chromosome 18. (A) Schematic diagram of the target region within chromosome 18 demarcating the position of the ZFN target site in the slc24a5 (golden) locus and the TALEN target site in the lepa locus. (B) Segmental deletions of 5.5 Mb are detected in normal-appearing nuclease-treated embryos when the ZFN and TALEN RNAs are coinjected. The presence or absence of each nuclease component (5′ and 3′ recognition unit) is indicated above each gel lane, as is the dose of each component. PCR products corresponding to the desired 5.5-Mb segmental deletion (413 bp) are detected only in embryos injected with both nucleases. The estimated deletion rate is indicated below the positive lane. (C) Junction sequences for the 5.5-Mb segmental deletions on chromosome 18. The nuclease recognition sequences are indicated above the junction sequences, where the half-sites are differentially colored. In the recovered junction sequences, there are variable length deletions as well as evidence of insertions between some of the target sites, where the latter are indicated in plain text.
