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Mammalian oocytes and zygotes are extraordinarily resilient receptacles that provide a conduit from designs etched in laboratory notebooks to living animals. The first transgenic mammals were generated three decades ago by injection of naked DNA into the pronuclei of mouse zygotes. Until now, however, pronuclear injection has allowed insertion of exogenous DNA only at random sites in the genome, and site-specific engineering has proved extremely difficult. In this issue, Cui and colleagues1 have finally overcome this barrier, making use of zinc-finger nucleases (ZFNs) to stimulate targeted integration of transgenes by homologous recombination in mouse and rat zygotes. This technology will dramatically alter the speed with which genetic alterations can be generated in a variety of mammalian species. Evidence that mouse pro-nuclei are endowed with the machinery to support homologous recombination emerged more than 20 years ago, but the efficiencies were too low to be practically useful2. Instead, the technology to manipulate the mouse genome has relied on embryonic stem (ES) cells, which are extraordinarily receptive to homologous recombination. Mouse ES cells with targeted mutations are now available for >12,000 genes; these cells have thus become the genetic repository for the mouse. Unfortunately, the link between cultured ES cells and the production of mutant animals could not be readily established in other species. ES cell lines isolated from species other than the mouse rarely exhibit germline colonization, although recent success has been reported in the rat3. The lack of authentic ES cells held back targeted genome manipulation in most mammalian species for many years. This barrier was eventually overcome by exploiting the remarkable ability of a mammalian oocyte to reprogram a somatic cell nucleus, effectively converting it to a zygotic genome (Fig. 1). By performing gene targeting in cultured somatic cells and then using the engineered cells for nuclear transfer, it became possible to manipulate endogenous genes in several mammalian species4. Despite these successes, the technical difficulties have been substantial because of the low efficiencies of both gene targeting in somatic cells and subsequent reprogramming of their nuclei. The research reported by Cui et al.1 provides for the first time a route to directly manipulate the rat genome, an approach that bypasses the requirement for germline competent ES cells or somatic cell nuclear transfer (Fig. 1). The major difficulty in achieving gene targeting with naked DNA injected into pro-nuclei is the very low efficiency of targeted rather than random integration2. Gene targeting is stimulated by several orders of magnitude in somatic cells by provision of a double-strand break in the genome5. Cleaving the mammalian genome at a defined site was not possible before the development of ZFNs, modular proteins that couple zinc-finger DNA-binding domains to the nuclease domain of the restriction endonuclease FokI6. They function as homo- or heterodimers, cleaving DNA between the two binding sites (Fig. 1), and in principle can be designed to target any unique site in complex genomes. After the initial demonstration of sequence-specific cleavage of the Drosophila melanogaster genome7, ZFNs were shown to stimulate targeted integration of a template sequence by means of homologous recombination in fruitflies, plants and human cells. Cui et al.1 and a recent report8 both demonstrate that co-injection of a pair of ZFN mRNAs with a targeting vector into pro-nuclei stimulates the frequency of gene targeting in mice to workable levels of 2–20% (Fig. 1). Cui et al.1 extend this approach to the rat. Interestingly, live-born founder animals obtained from these experiments1, 8 are mosaics that carry several different mutant alleles with deletions at the target locus, as well as correctly targeted alleles and unmodified wild-type alleles. Deletions are expected products following nonhomologous end-joining of cleaved DNA in the absence of targeting and have been described previously following expression of ZFNs in zygotes9. The transmission of multiple different mutant alleles from the same founder reflects germline mosaicism caused by expression and cleavage activity of ZFNs after DNA replication in maternal and/or paternal pro-nuclei. The ability to simultaneously generate a spectrum of mutations can be advantageous for genetic purposes. Despite these advances, several questions about ZFN-stimulated pronuclear targeting remain to be addressed. Molecular biologists are familiar with unwanted off-target activity of restriction enzymes. To what extent do ZFNs cleave other sites in the genome? The comparatively small size of deletions generated at illegitimate sites suggests that off-target cleavages will be hard to trace. Does the physical damage of the host genome observed in many transgenic animals generated by pronuclear injection occur in this setting, too? The importance of the answers to these questions will undoubtedly depend on the frequency and type of unwanted events, their linkages to the desired genomic alterations and the context in which the technology is applied. The mutant alleles generated using conventional gene targeting technology in mouse ES cells have become increasingly sophisticated over the past two decades. The repertoire of genetic alterations that can be achieved by ZFN-stimulated pronuclear targeting is fertile ground for exploration. Although mutant rats have recently been produced using rat ES cell technology10 (Fig. 1), ZFN targeting applied directly to the zygote (Fig. 1) presents considerable advantages. Pronuclear injection of nucleic acids is well established, widely practiced and applicable to any strain. Moreover, transmission of the engineered allele from founder rats is readily achieved. Provided that ZFNs with the appropriate specificity can be generated, the community of rat researchers can look forward to rats with a myriad of defined genome modifications. This technology will also find applications in a multitude of other species, which hitherto have required somatic cell reprogramming to achieve directed modifications of their genomes. Although vector-chromosome gene targeting has yet to be demonstrated in the pro-nuclei of farm animal zygotes, this is likely to be possible. ZFNs have been shown to stimulate gene targeting in a variety of species and can therefore be used in combination with somatic nuclear transfer, removing a bottleneck in achieving directed modification by this route. The promise of this technology will stimulate numerous applications, but the terms, conditions and costs associated with commercially provided ZFNs can be prohibitive and may limit their potential. Download references

Crafting rat genomes with zinc fingers