Design and Testing of Zinc Finger Nucleases for Use in Mammalian Cells

Homologous recombination is the most precise way to manipulate the genome. As a tool it has been used extensively in bacteria, yeast, murine embryonic stem cells, and a few other specialized cell lines but has not been available to researchers in other systems, such as for mammalian somatic cell genetics. Recently, work has shown that the creation of a gene-specific DNA double-strand break can stimulate homologous recombination by several thousand-fold in mammalian somatic cells. These double-strand breaks can now be created in mammalian genomes by zinc finger nucleases (ZFNs). ZFNs are artificial proteins in which a zinc finger DNA-binding domain is fused to a nonspecific nuclease domain. This chapter describes how to identify potential targets for ZFN cutting, to make ZFNs to cut this target site, and how to test whether the newly designed ZFNs are active in a mammalian cell culture-based system.