In the last 20 years, the availability of knockout mouse technology has established the mouse as the most widely used model system in biomedical research. The key enabling aspect of knockout mouse technology lies in the ease with which mouse embryonic stem (mES) cells can be cultured, genetically modified, cloned, and implanted into blastocysts to derive chimeric animals.
In particular, mES cells are relatively amenable to targeted genetic modification using HR and selectable markers while other cell types are not. For instance, large efforts have been mounted to establish a similar knockout rat approach by focusing on the culture of rat ES cells. However, while this effort culminated in the successful isolation of rat ES cells in 2008, gene-targeting techniques have not been developed, underlining the need for alternative genome manipulation techniques.
In 2009, the first targeted knockout rats were created via microinjection of ZFNs into single-cell embryos, resulting from a collaboration involving Sigma-Aldrich, Sangamo BioSciences, the Medical College of Wisconsin, OMT, and INSERM. The gene knockout process relied on the ability of ZFNs to create high frequency insertions and deletions through the DNA repair process of NHEJ.
The method for delivering ZFNs as capped poly-tailed mRNA via embryo microinjection and screening of subsequent animals was rapid and efficient, enabling the production of founders in as little as two months post-microinjection by screening less than 100 animals. Independent follow-up efforts have produced IL2Rγ knockout rats.
Most importantly, the method by which the ZFN-derived knockout rat was generated suggests a general solution to targeted gene knockout in other species. ZFN-mediated gene knockout does not require the establishment of ES cell culture for the species of interest, but simply requires that embryo isolation, injection, and implantation protocols be well characterized.
Many animal species have embryo-manipulation methods robust enough to support the creation of transgenic animals, including mice, rats, rabbits, chickens, sheep, goats, cows, and pigs. The same methods used to create transgenic animals can be used to efficiently deliver ZFNs to embryos and potentially derive knockout animals. Even when ES cell culture is established for a given species, ZFNs offer a faster route to modified animals by manipulating the embryo directly without the need for assembling targeting plasmids required for gene knockout in ES cells (Figure 2).