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Lentiviral Vectors Encoding Zinc-Finger Nucleases Specific for the Model Target Locus HPRT1

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Gene Correction

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1114))

Abstract

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.

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Acknowledgments

The authors thank Dr. Antoine A.F. de Vries (Department of Cardiology, LUMC, The Netherlands) for technical assistance in setting up the PEI transfection protocol and Ignazio Maggio (Department of Molecular Cell Biology, LUMC, The Netherlands) for generating the ZFN-encoding lentiviral vector transfer plasmids. We also thank Prof. Rob Hoeben (Department of Molecular Cell Biology, LUMC, The Netherlands) for his critical reading of the manuscript. This work was supported by the Prinses Beatrix Spierfonds (grant W.OR11-18) and by the European Community’s 7th Framework Programme for Research and Technological Development: PERSIST-Persisting Transgenesis (grant agreement number 222878).

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Authors and Affiliations

  1. Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands

    Laetitia P. L. Pelascini & Manuel A. F. V. Gonçalves

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  1. Laetitia P. L. Pelascini
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  2. Manuel A. F. V. Gonçalves
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Editors and Affiliations

  1. Georgia Institute of Technology, Atlanta, Georgia, USA

    Francesca Storici

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Pelascini, L.P.L., Gonçalves, M.A.F.V. (2014). Lentiviral Vectors Encoding Zinc-Finger Nucleases Specific for the Model Target Locus HPRT1 . In: Storici, F. (eds) Gene Correction. Methods in Molecular Biology, vol 1114. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-761-7_12

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  • DOI: https://doi.org/10.1007/978-1-62703-761-7_12

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-760-0

  • Online ISBN: 978-1-62703-761-7

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