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Screening Strategies for TALEN-Mediated Gene Disruption

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Programmed Cell Death

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1419))

Abstract

Targeted gene disruption has rapidly become the tool of choice for the analysis of gene and protein function in routinely cultured mammalian cells. Three main technologies capable of irreversibly disrupting gene-expression exist: zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. The desired outcome of the use of any of these technologies is targeted insertions and/or deletions (indels) that result in either a nonsense frame shift or splicing error that disrupts protein expression. Many excellent do-it-yourself systems for TALEN construct assembly are now available at low or no cost to academic researchers. However, for new users, screening for successful gene disruption is still a hurdle. Here, we describe efficient and cost-effective strategies for the generation of gene-disrupted cell lines. Although the focus of this chapter is on the use of TALENs, these strategies can be applied to the use of all three technologies.

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Acknowledgments

We thank Mike Ryan, Michael Lazarou, Rochelle Tixeira, and Thanh N. Nguyen for reagents, advice, and discussions. This work was supported by the Australian National Health and Medical Research Council (NHMRC fellowship 1070916 to DAS) and the Australian Mitochondrial Disease Foundation (AMDF).

Author information

Authors and Affiliations

  1. The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia

    Boris Reljić

  2. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia

    Boris Reljić

  3. Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Wellington Road, Clayton 3800, Melbourne, VIC, Australia

    David A. Stroud

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  1. Boris Reljić
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  2. David A. Stroud
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Correspondence to David A. Stroud .

Editor information

Editors and Affiliations

  1. Department of Biochemistry and Genetics, La Trobe Inst for Mol Sci, La Trobe Univ, Bundoora, Victoria, Australia

    Hamsa Puthalakath

  2. Department of Biochemistry and Genetics, La Trobe Inst for Mol Sci, La Trobe Univ, Bundoora, Victoria, Australia

    Christine J. Hawkins

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Reljić, B., Stroud, D.A. (2016). Screening Strategies for TALEN-Mediated Gene Disruption. In: Puthalakath, H., Hawkins, C. (eds) Programmed Cell Death. Methods in Molecular Biology, vol 1419. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3581-9_17

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  • DOI: https://doi.org/10.1007/978-1-4939-3581-9_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3579-6

  • Online ISBN: 978-1-4939-3581-9

  • eBook Packages: Springer Protocols

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