Analysis of Targeted Chromosomal Deletions Induced by Zinc Finger Nucleases

Abstract

INTRODUCTIONTargeted genomic deletions of large DNA segments in higher eukaryotic cells and organisms have been difficult, if not impossible, to achieve. Current approaches for targeted genomic deletions utilizing recombination systems (such as Flp/FRT and Cre/loxP) or bacterial artificial chromosomes are mostly limited to mouse embryonic stem cells, which support genetic manipulation by homologous recombination. Zinc finger nucleases (ZFNs) are artificial restriction enzymes, made by fusing an engineered zinc finger DNA-binding domain (which targets a desired sequence) to the DNA cleavage domain of the FokI type IIS restriction enzyme. In the cell, ZFNs can generate targeted DNA double-strand breaks on a chromosome and thus have been used to introduce site-specific, local mutations to a gene of interest. In addition, ZFNs have been recently reported to induce large genomic deletions in cultured human cells. In this protocol, we describe methods to detect and analyze large genomic deletions in cultured cells introduced by expression of ZFNs that target two separate sites on a chromosome.