The construction and expression of ZFNs.

<p>(A) Three-step PCR for the construction of DNA-binding domain of ZFs. PCR products for each PCR step for 6-finger ZF (left-ZF of <i>Rosa26</i>), 5-finger ZF (left-ZF of <i>Il2rg</i>), and 4-finger ZF (left-ZF of <i>Gli3</i>) are shown. Arrows indicate the intended base pairs of PCR products. (B) Construction efficiencies of left- and right-ZFNs for 4 target loci. (C, D) The expression of constructed ZFN in mouse zygotes. ZFN mRNA at 200 µg/ml (100 µg/ml each for right- and left-ZFNs) were injected into the cytoplasm of mouse zygotes and after 4 h, ZFN protein expression was confirmed by immunoblotting (C) and immunocytochemistry (D) with anti-Flag antibody. Alpha-tubulin immunoblotting is shown as the internal control, and DNA was stained by propidium iodide.