Testing a Three-Finger Zinc Finger Nuclease Using a GFP Reporter System
Adapted from Gene Transfer: Delivery and Expression of DNA and RNA (ed. Friedmann and Rossi). CSHL Press, Cold Spring Harbor, NY, USA, 2007.INTRODUCTION
Homologous recombination is the most precise way to manipulate the genome. It has been used extensively in bacteria, yeast, murine embryonic stem cells, and a few other specialized cell lines, but it has not been available in other systems such as mammalian somatic cells. However, the creation of a gene-specific DNA double-strand break can stimulate homologous recombination by several-thousandfold in mammalian somatic cells. These double-strand breaks can be created in mammalian genomes by zinc finger nucleases (ZFNs), artificial proteins in which a zinc finger DNA-binding domain is fused to a nonspecific nuclease domain. This protocol describes how to test newly designed ZFNs using a cell-based green fluorescent protein (GFP) reporter assay to determine if they are active in a mammalian cell-culture-based system.
