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Targeted gene addition to mammalian genomes is central to biotechnology, basic research and gene therapy. For example, gene targeting to the ROSA26 locus by homologous recombination in embryonic stem cells is commonly used for mouse transgenesis to achieve ubiquitous and persistent transgene expression. However, conventional methods are not readily adaptable to gene targeting in other cell types. The emerging zinc finger nuclease (ZFN) technology facilitates gene targeting in diverse species and cell types, but an optimal strategy for engineering highly active ZFNs is still unclear. We used a modular assembly approach to build ZFNs that target the ROSA26 locus. ZFN activity was dependent on the number of modules in each zinc finger array. The ZFNs were active in a variety of cell types in a time- and dose-dependent manner. The ZFNs directed gene addition to the ROSA26 locus, which enhanced the level of sustained gene expression, the uniformity of gene expression within clonal cell populations and the reproducibility of gene expression between clones. These ZFNs are a promising resource for cell engineering, mouse transgenesis and pre-clinical gene therapy studies. Furthermore, this characterization of the modular assembly method provides general insights into the implementation of the ZFN technology.

Gene targeting to the ROSA26 locus directed by engineered zinc finger nucleases