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Use of RNA Interference to Study DNA Repair

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Genotoxicity and DNA Repair

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

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Abstract

DNA repair pathways maintain the integrity of the genome and thereby help prevent the onset of cancer, disease, and aging phenotypes [DNA Repair and Mutagenesis, ASM, Washington, DC]. As such, the critical requirement for DNA repair proteins and pathways in response to radiation and genotoxic chemotherapeutics implicates DNA repair proteins as prime targets for improving response to currently available anticancer regimens. Although defects in critical DNA repair pathways or proteins can predispose to cancer onset [FASEB J 26:2094–2104, 2012], such cancer-specific DNA repair defects offer novel approaches for tumor-selective therapy.

To effectively evaluate the functional role of a specific DNA repair protein with regard to cell survival, response to genotoxins, and genome stability, it has become standard practice to employ select genetic tools to alter expression of the gene of interest and/or reexpress a mutant transgene [Cancer Res 71:2308–2317, 2011; Mol Cancer Res 8:67–79, 2010; Neuro Oncol 13:471–486, 2011]. A useful approach to reduce or suppress a specific gene of interest in cells is RNA interference. Briefly, RNA interference is a posttranscriptional gene-silencing biological mechanism whereby RNA molecules inhibit gene expression either by translational suppression or by the targeted degradation of specific mRNA molecules. Once the gene of interest is suppressed in this manner and validated for gene expression loss, the resulting knockdown (KD) cells can be used for functional analysis to define the cellular impact of gene loss and provide a resource for evaluating mutants of the gene, such as somatic or germ-line mutations, for impact on function [PLoS Genet 8:e1003086, 2012]. Herein, we describe methods to modify human cells via RNA interference as well as methods to validate gene KD and some measures of cellular response to genotoxins to uncover functional DNA repair defects in the absence of the gene.

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Acknowledgments

This work was supported by grants from the National Institute of Health (NIH) (CA148629, GM087798, and GM099213) to R.W.S. Support for the UPCI Lentiviral Facility was provided by the Cancer Center Support Grant from the National Institutes of Health (CA047904).

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Authors and Affiliations

  1. Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA

    Elise Fouquerel, Jianfeng Li, Andrea Braganza, David Svilar, Qingming Fang & Robert W. Sobol Ph.D.

  2. Hillman Cancer Center, University of Pittsburgh Cancer Institute, Research Pavilion, Suite 2.6a, 5117 Centre Avenue, Pittsburgh, PA, 15213-1863, USA

    Elise Fouquerel, Jianfeng Li, Andrea Braganza, Zhongxun Yu, Ashley R. Brown, Xiao-Hong Wang, Sandy Schamus, David Svilar, Qingming Fang & Robert W. Sobol Ph.D.

  3. School of Medicine, Tsinghua University, No. 1 Tsinghua Yuan, Haidian District, Beijing, 100084, China

    Zhongxun Yu

  4. Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, 15213, USA

    Robert W. Sobol Ph.D.

Authors
  1. Elise Fouquerel
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  2. Jianfeng Li
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  4. Zhongxun Yu
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  5. Ashley R. Brown
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  6. Xiao-Hong Wang
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  7. Sandy Schamus
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  8. David Svilar
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  9. Qingming Fang
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  10. Robert W. Sobol Ph.D.
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Correspondence to Robert W. Sobol Ph.D. .

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Editors and Affiliations

  1. Universidad de Oviedo, Oviedo, Spain

    L. María Sierra

  2. Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal

    Isabel Gaivão

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Fouquerel, E. et al. (2014). Use of RNA Interference to Study DNA Repair. In: Sierra, L., Gaivão, I. (eds) Genotoxicity and DNA Repair. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1068-7_24

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  • DOI: https://doi.org/10.1007/978-1-4939-1068-7_24

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1067-0

  • Online ISBN: 978-1-4939-1068-7

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