Targeted integration in rat and mouse embryos with zinc-finger nucleases

Abstract

Cui et al. generate transgenic rats and mice bearing targeted genomic integrations by enhancing the rate of homologous recombination in single-cell embryos with zinc-finger nucleases. The approach avoids the time-consuming backcrossing involved in generating mutant mice with ES cells and should be applicable to species for which ES cells have not been isolated. Gene targeting is indispensible for reverse genetics and the generation of animal models of disease. The mouse has become the most commonly used animal model system owing to the success of embryonic stem cell–based targeting technology1, whereas other mammalian species lack convenient tools for genome modification. Recently, microinjection of engineered zinc-finger nucleases (ZFNs) in embryos was used to generate gene knockouts in the rat2,3 and the mouse4 by introducing nonhomologous end joining (NHEJ)-mediated deletions or insertions at the target site. Here we use ZFN technology in embryos to introduce sequence-specific modifications (knock-ins) by means of homologous recombination in Sprague Dawley and Long-Evans hooded rats and FVB mice. This approach enables precise genome engineering to generate modifications such as point mutations, accurate insertions and deletions, and conditional knockouts and knock-ins. The same strategy can potentially be applied to many other species for which genetic engineering tools are needed.