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REVIEW.com/naturebiotechnologyGene targeting using zinc finger
nucleaseshttp://www.nature2005 Nature Publishing Group Matthew H Porteus1 & Dana Carroll2The ability to achieve site-specific manipulation of the mammalian genome has widespread implications for
basic and applied research. Gene targeting is a process in which a DNA molecule introduced into a cell replaces
the corresponding chromosomal segment by homologous recombination, and thus presents a precise way to
manipulate the genome. In the past, the application of gene targeting to mammalian cells has been limited by
its low efficiency. Zinc finger nucleases (ZFNs) show promise in improving the efficiency of gene targeting by
introducing DNA double-strand breaks in target genes, which then stimulate the cells endogenous homologous
recombination machinery. Recent results have shown that ZFNs can be used to create targeting frequencies of up
to 20% in a human disease-causing gene. Future work will be needed to translate these in vitro findings to in vivo
applications and to determine whether zinc finger nucleases create undesired genomic instability.Knowledge of the complex interplay between the genome, the physiologic processes it governs and the environment with which it interacts
has increased. Gene targeting has provided an important research tool
for probing this complex interplay and for manipulating the genome.
In gene targeting, an exogenously introduced DNA fragment replaces
an endogenous segment of DNA by homologous recombination
(Box 1). This process was reported in yeast more than 25 years ago and
variations on this technique are now commonly used for evaluating
gene function in that organism13.Gene targeting also has been demonstrated in mouse cells, and
when it is applied to embryonic stem (ES) cells, it has enabled the
production of mutant mice, both for studying gene function and for
creating models of human genetic diseases4,5. Although positive selection for the integrated gene is sufficient to recover the desired yeast
cells, elegant and powerful selections for the transgene and against
nontargeted integration had to be devised for gene targeting in mouse
cells where most of the transgenes integrate at inappropriate sites in
the genome; the advances have made this technique nearly routine6,7.
Thousands of transgenic mice and ES cell lines with precise genomic
alterations have been created; their characterization has increased
our understanding of mammalian physiology and the pathogenesis
of numerous human diseases. Nonetheless, many applications of...