Generating a transgenic or knockout mouse is a labor-intensive, long-term project often requiring years of experiments and crosses. Although these approaches work, the slow pace of the traditional methodology cannot keep up with the tidal wave of genetic variants being identified by high-throughput sequencing.
Previously, genetically modified mice have been generated by introducing mutations into mouse embryonic stem (ES) cells and then injecting those cells into a blastocyst. That blastocyst is then implanted into a surrogate mother mouse who births chimeric mice that contain cells with different genotypes. But because only some of those chimeras have mutant cells in their germline tissues, not all of the resulting mice can pass the modified genotype to offspring—a multigenerational endeavor.
Building on their previous work using zinc-finger nucleases (2), Wurst and his team took advantage of another class of nuclease-based engineering tools known as transcription activator-like effector nucleases (TALENs) to introduce targeted genetic modifications directly into one-celled mouse embryos. “We tried it with zinc fingers and it worked, so the logical extension was then with TALENs. You can design TALENs, you can find many TALEN target sites, and they are relatively easy to produce. You can use oligonucleotides [for targeting]. You don’t need to have a sophisticated vector,” explained Wurst.
The real benefit though is that you don’t need to produce chimeras and screen for germline transmission. “Basically one afternoon session of injections gives you enough founders to obtain the mutations you would like to have. To be honest, we were truly surprised how well it works,” said Wurst.
The mice produced by this method express the altered genotype in every cell. As a result, heterozygotes can be produced in 18 weeks and homozygotes in 25 weeks. Wurst and his team have even produced homozygous mutants, albeit at a low rate, by genetically modifying a single one-celled embryo.
Such direct genetic manipulation of embryos allows researchers to create more genetic variability, which Wurst believes will advance understanding of disease genetics. “You can make very fast point mutations. We believe point mutations are the next important step in validating genetic variants that have been found in human conditions. You would like to have as many point mutations as you can in disease models.”
As for the future, Wurst sees more potential in this approach. “I believe there can be additional improvements here—these are more or less our first trials.”
1. Wefers B, Meyer M, Ortiz O, Hrabé de Angelis M, Hansen J, Wurst W, Kühn R. 2013. Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides. Proc Natl Acad Sci U S A. 110(10):3782-7.
2. Meyer M, de Angelis MH, Wurst W, Kühn R. 2010. Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci U S A .107(34):15022-6.