Experimental schemes for vector construction.

<p>(A) Composition of the platform vector used for ZFN construction. (B) Construction procedures for ZFN vectors by OLTA. In this figure, construction of a DNA-binding domain composed of 4 ZFs is shown as an example. Three partial ZF fragments were synthesized by the 1st PCR with the primer sets shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059801#pone-0059801-t002" target="_blank">Table 2</a> and they were combined by overlap extension PCR (2nd PCR) and amplified by 3rd PCR; then the DNA-binding domain was joined with the platform vector by TA cloning. (C) A scheme showing constructs of <i>in vitro</i> transcribed ZFN mRNA and <i>in vivo</i> translated ZFN protein, and ZFN binding with target DNA; <i>Gli3</i> target site is shown in this figure for example.