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.
The Tokunaga Laboratory, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita, 879-5593, Japan.|Section of Physiology, Department of Integrative Aging Neuroscience, National Center for Geriatrics and Gerontology (NCGG), 7-430, Morioka-cho, Obu, Aichi, 474-8511, Japan.
Akinori
A
Tokunaga
Clinical Engineering Research Center, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita, 879-5593, Japan.
Hirofumi
H
Anai
Clinical Engineering Research Center, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita, 879-5593, Japan. hanada@oita-u.ac.jp.
Katsuhiro
K
Hanada
D053903:DNA Breaks, Double-Stranded; D003851:Deoxyribonucleases / Q000737:chemistry / Q000502:physiology; D056890:Eukaryota / Q000235:genetics; D018390:Gene Targeting / Q000379:methods* / Q000639:trends; D059765:Homologous Recombination; D008957:Models, Genetic; D015202:Protein Engineering
D003851:Deoxyribonucleases
Designer nucleases; Double-stranded DNA breaks; Homologous recombination