Advances in stem-cell technology have broken the barrier to gene targeting in mammals other than mice. A wide array of research opportunities now opens up, especially in studies involving the laboratory rat.
Ever since the 1980s, and the Nobel prizewinning work of Capecchi, Evans and Smithies1, gene-targeting technology has been applied to embryonic stem (ES) cell cultures, although its use has been limited to mice. This is now no longer the case. In little more than a year, three publications2,3,4 have reported new avenues for gene targeting in a second mammalian species, the laboratory rat — the rat being the most widely used model organism for deciphering how human organ systems function, and for testing medicines. The latest of these reports, by Tong et al.4, appears on page 211 of this issue.
Specific alteration of the genetic make-up of organisms including plants, yeast, flies and mice by gene targeting is the most powerful way to study how genes govern biological processes. Different targeting methods allow individual genes to be added (knocked in), disrupted (knocked out) or modified in DNA content at their specified locus within a genome. This procedure allows direct comparisons of the disease susceptibility of animals inheriting a targeted mutation with the susceptibility of normal animals (or plants), so providing the genetic evidence for testing biological hypotheses or defining biochemical targets for new pharmaceuticals.
In the first of the three publications, Geurts and colleagues2 described how they microinjected specially designed enzymes, called site-specific nucleases, into one-cell embryos to disrupt target genes in rats. Site-specific nucleases (zinc-finger nucleases) are engineered in the lab as 'molecular scissors' that snip out small parts of a target gene from an organism's genome. In a second approach, Izsvák et al.3 disrupted a battery of 35 rat genes by transposon mutagenesis in cultures of sperm stem cells. Transposons are nature's own version of small mutagenic DNA elements, which can be coaxed to 'hop' into genes to affect their expression. Now comes the paper by Tong and colleagues4. They describe successful gene targeting in rat ES cells to disrupt expression of a well-known gene called p53.
The study by Tong et al.4 carries added significance because the authors disrupted p53 by gene replacement. Gene replacement is the most sophisticated method for gene targeting. This is due to its exquisite specificity and fidelity in exchanging genetic information at a locus of choice among the billions of DNA base pairs comprising a genome. Gene replacement uses a cell's natural DNA-repair process, homologous recombination, which allows genes designed by the researcher to replace a cell's own genes. Moreover, Tong et al. worked with cultures of ES cells, which have traits that are ideal for gene replacement. They are pluripotent, which means that they can give rise to almost all cell types in a developing organism when microinjected back into an early embryo. Also, ES cells divide efficiently in culture so that rare gene-replacement events (<1 in 10,000 cells receiving the test DNA) can be selected for and amplified in number within 'clones' of targeted cells for embryo injection.
So what's been the hold-up in achieving gene replacement in rats? To alter the genetic make-up of an entire animal, gene modifications must be permanently (stably) introduced into its germ-cell lineage. Embryonic germ cells derived from pluripotent ES cells develop into either eggs or sperm. Thus, mutated genes can be stably passed from ES cells into the genome of a newly formed organism by fertilization. This event is described as germline transmission of an individual's genome. Culture conditions for mouse ES cells have been finely honed over the past 30 years to optimize the cells' potential to enter the germ-cell lineage after being microinjected back into an early embryo. Accordingly, gene replacement in ES cells has been especially successful for producing mutant mice.
Therein lies the problem: culture conditions for growing germline-competent ES cells of mammals other than mice have long been a challenge. If both donor ES cells and their recipient embryo function properly, a surrogate mother carrying the constructed embryo will give birth to chimaeric animals — 'mosaics', composed of cell types derived from both the cultured ES cells and the recipient embryo. If a percentage of a chimaera's sperm or eggs are derived from injected ES cells, targeted genes are transmitted to a similar percentage of their offspring to make pure mutant strains. All cells in ES-cell-derived offspring have one copy of the targeted gene and one normal, unmutated copy of that gene (that is, they are pure mutant heterozygotes). Mating between two such heterozygotes will then produce some 25% of offspring that are homozygotes, that is, they have two copies of the targeted gene. If the mutation is disruptive, it can completely extinguish (knock out) expression of the gene.
However, if not cultured correctly, even mouse ES cell lines rapidly accumulate chromosomal abnormalities and/or develop more towards other cell lineages, such as those of nerves or muscle. These factors reduce the degree of germline chimaerism obtained in animals and, therefore, reduce the likelihood that the gene targets of donor ES cells will be transmitted to subsequent generations.
The gene-targeting success of Tong et al.4 rested on advances in cultivating germline-competent rat ES cells, which only in the past two years or so have been used to generate animals displaying germline chimaerism5,6,7. These advances were themselves precipitated by work that culminated in an eye-opening publication8 on the use of small-molecule inhibitors to block biochemical pathways that normally stimulate loss of ES-cell germline competency. The approach resulted in high levels of germline chimaerism in mice, and was swiftly adapted to produce chimaeric rats5,6.
All the evidence indicates that gene targeting in rats will become commonplace (Fig. 1). Site-specific nucleases are already being applied with great success to disrupt genes in animals, producing high rates of germline chimaerism. If improvements increase the fidelity of these nucleases for a wider spectrum of genomic sites, the application should increase proportionately. Although they are mainly used to disrupt genes, site-specific nucleases also mobilize a cell's homologous-recombination machinery by catalysing DNA breaks within a target gene9. This property makes them, and transposons, highly attractive as co-factors for enhancing normally low rates of gene replacement at a target locus. Such a dual approach could further revolutionize genetics if it is optimized for routinely replacing genes in oocytes9, early embryos or germline-competent stem cells of various species.
If the level of success in targeting rat p53 is representative of most genes, a door has now been opened to a bright future in rat genetics, with — we may hope — other species to follow. Gene replacement is currently the only method by which one can insert elaborate DNA sequences into a specific genetic locus. It dramatically increases the options of how a locus can be experimentally designed in animals to answer key biological questions. In the long term, after most of the tens of thousands of known genes of any given species have been disrupted at a fundamental level by various gene-targeting approaches, endless applications for gene replacement to more elegantly modify its genome are likely to ensue.
Looking beyond the reliance on early embryos and ES cells for gene targeting, cultures of sperm stem cells (developmentally termed spermatogonia) offer a promising route for further innovation in animal genetics. It has been shown in mice10 that injecting sperm stem cells into testes assures germline transmission of replaced genes while providing a more streamlined route to making pure mutant animals. Optimizing the sperm stem-cell approach in other species will offer the advantage that it does not require breeding schemes to screen embryo-derived chimaeras for germline transmission. Given the long duration of most species' reproductive cycles, untold time would thus be saved in producing new strains.
Geurts, A. M. et al. Science 325, 433 (2009).
Izsvák, Z. et al. Nature Methods 7, 443–445 (2010).
Tong, C., Li, P., Wu, N. L., Yan, Y. & Ying, Q.-L. Nature 467, 211–213 (2010).
Buehr, M. et al. Cell 135, 1287–1298 (2008).
Li, P. et al. Cell 135, 1299–1310 (2008).
Kawamata, M. & Ochiya, T. Proc. Natl Acad. Sci. USA 107, 14223–14228 (2010).
Ying, Q.-L. et al. Nature 453, 519–523 (2008).
Bibikova, M. et al. Mol. Cell. Biol. 21, 289–297 (2001).
Kanatsu-Shinohara, M. et al. Proc. Natl Acad. Sci. USA 103, 8018–8023 (2006).
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