Assembly and in vitro Functional Analysis of Zinc Finger Nuclease Specific to the 3′ Untranslated Region of Chicken Ovalbumin Gene

Synthetic zinc finger nucleases (ZFNs) are useful for the improvement of site directed integration of foreign gene into vertebrate chromosomes. To facilitate site-directed integration of foreign genes into the 3'-untranslated region of the chicken ovalbumin gene, we have constructed ZFN expression vectors using Zinc Finger Consortium Vector Kits and tested the functionality of these ZFN constructs. Coding sequences for 6 zinc fingers were assembled following the modular assembly method. The zinc finger assembly was fused to two FokI catalytic domains. Various configurations of linker regions between domains were tested for their influence on enzymatic activity, using plasmid substrate containing the target sequence. Results indicated that ZFN with an elongated linker between two nuclease domains had a high catalytic activity.