Mechanisms of gene targeting in higher eukaryotes

Targeted genome modifications using techniques that alter the genomic information of interest have contributed to multiple studies in both basic and applied biology. Traditionally, in gene targeting, the target-site integration of a targeting vector by homologous recombination is used. However, this strategy has several technical problems. The first problem is the extremely low frequency of gene targeting, which makes obtaining recombinant clones an extremely labor intensive task. The second issue is the limited number of biomaterials to which gene targeting can be applied. Traditional gene targeting hardly occurs in most of the human adherent cell lines. However, a new approach using designer nucleases that can introduce site-specific double-strand breaks in genomic DNAs has increased the efficiency of gene targeting. This new method has also expanded the number of biomaterials to which gene targeting could be applied. Here, we summarize various strategies for target gene modification, including a comparison of traditional gene targeting with designer nucleases.