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Micro and Nano Technologies in Bioanalysis pp 617–636Cite as

Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 544))

Summary

Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity.

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Acknowledgments

We thank Dr. Sundar Durai for drawing the figures. The research on ZFNs in our lab has been supported by various grants from National Institutes of Health, USA, during the past 13 years; it is currently being funded by the research grant GM077291 from NIGMS/NIH. Our work on ZFN-mediated gene targeting in human stem cells is partially supported by a grant from the Maryland Stem Cell Research Fund.

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Authors and Affiliations

  1. Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, 21205, Baltimore, MD, USA

    Karthikeyan Kandavelou & Srinivasan Chandrasegaran

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  1. Karthikeyan Kandavelou
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  2. Srinivasan Chandrasegaran
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Correspondence to Srinivasan Chandrasegaran .

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    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

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    Kandavelou, K., Chandrasegaran, S. (2009). Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes. In: Foote, R., Lee, J. (eds) Micro and Nano Technologies in Bioanalysis. Methods in Molecular Biology™, vol 544. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-483-4_40

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    • DOI: https://doi.org/10.1007/978-1-59745-483-4_40

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    • Publisher Name: Humana Press, Totowa, NJ

    • Print ISBN: 978-1-934115-40-4

    • Online ISBN: 978-1-59745-483-4

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