Engineering and applications of zinc finger nucleases.

摘要

Zinc finger nucleases (ZFNs) that combine the zinc finger proteins (ZFPs) with the non-specific cleavage domain of FokI restriction enzyme are becoming powerful tools in the repertoire of molecular biologists for genome engineering of plant and mammalian cells including the human cells. ZFN-mediated gene targeting has been used for gene editing and directed mutagenesis of specific sites within plant and mammalian genome. In this thesis, I report the design, engineering and rapid in vitro characterization of the sequence specific cleavage properties of three-finger ZFNs that target specific sites within the mouse tyrosinase (mTYR) and mouse cystic fibrosis transmembrane conductance regulator (mCFTR) genes, respectively. Since the engineering and characterization of ZFNs to target a specific gene is a lengthy process, the rapid protocol reported here should promote widespread use of ZFN-mediated gene targeting for genome engineering of plants and mammalian cells.; Since continued expression of ZFNs has proven to be harmful to cells, I have developed a Tet-Off expression system for regulated expression of the designed mTYR ZFNs in albino mouse melanocytes. This system will allow for a systematic comparison of the efficiency of ZFN-mediated gene targeting versus ZFNs cytotoxicity within cells. I have initiated studies on ZFN-mediated gene editing of the mTYR gene in albino mouse melanocytes under regulated expression of the designed ZFNs. I have also initiated studies to investigate if the efficiency of ZFN-mediated gene targeting can be improved by coupling it with a positive-negative selection approach.; I have used the biotin pull-down assay to show that ZFN fusion proteins, like FokI endonuclease, form dimers only when ZFN monomers are bound to specific sites. The physical association of two ZFN monomers was confirmed in my study and it gives us a better understanding about the mechanism of double-strand cleavage by ZFNs. I have also shown that some of the previously constructed ZFN variants, which included more of the FokI domains to regulate the cleavage domain, were also active and that they also form dimers.; My thesis work addresses some of the challenges that face ZFN-mediated gene targeting before its full potential can be realized and it can be considered for human therapeutics as a form of gene therapy.