TALEN-generated 39-kb and 69-kb deletions within the zebrafish flt4 locus. (A) Schematic diagram of the flt4 locus demarcating the positions of all three TALEN binding sites and the distance separating them. (B) Segmental deletions of 39 and 69 kb are readily detected in normal-appearing TALEN-treated embryos when two TALENs are coinjected. The presence or absence of each TALEN component (5′ and 3′ recognition unit) is indicated above each gel lane, as is the dose of each TALEN component. PCR products corresponding to the desired 39-kb and 69-kb segmental deletion products are detected only in embryos injected with both TALENs. Estimated deletion rates are indicated below the positive lanes. Segmental inversions are detected in embryos treated with two TALENs for both segments, but at lower rates than the corresponding segmental deletions. Junction sequences for the 39-kb (C) and 69-kb (D) segmental deletions at the flt4 locus. The TALEN-1 and TALEN-2/3 recognition sequences are indicated above the junction sequences, where the half-sites recognized by each TALEN 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.
