Lentiviral vectors encoding zinc-finger nucleases specific for the model target locus HPRT1

Zinc-finger nucleases (ZFNs) are artificial proteins designed to induce double-stranded DNA breaks (DSBs) at predefined chromosomal positions. These site-specific genomic lesions facilitate the study of translocations and cellular DNA repair processes and serve as powerful stimuli for the editing of complex genomes. The delivery of ZFNs into a wide range of cell types is of utmost importance for the broad evaluation and deployment of the technology. Lentiviral vectors (LVs) are versatile gene delivery vehicles that transduce alike transformed and primary cells regardless of their division rate. In this chapter, we describe the generation of conventional and integrase-defective LVs encoding ZFNs targeting the human hypoxanthine phosphoribosyltransferase 1 (HPRT1) locus. Furthermore, we introduce a general LV titration method based on a cost-effective quantitative PCR protocol and implement a rapid and simple restriction enzyme site polymorphism assay to detected DSB formation at the HPRT1 target sequence. Owing in part to the small molecule-based clone selection schemes conferred by HPRT1 allelic knockouts, this X-linked gene has become a "classical" target model locus in mammalian cells. The reagents and techniques detailed herein yield LV preparations that induce HPRT1-specific DSBs. As a result, they should constitute a valuable resource to increase the robustness and decrease the timelines of the various protocols based on HPRT1 gene disruption and targeting.