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Increased Gene Targeting in Ku70 and Xrcc4 Transiently Deficient Human Somatic Cells

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Abstract

The insertion of foreign DNA at a specific genomic locus directed by homologous DNA sequences, or gene targeting, is an inefficient process in mammalian somatic cells. Given the key role of non-homologous end joining (NHEJ) pathway in DNA double-strand break (DSB) repair in mammalian cells, we investigated the effects of decreasing NHEJ protein levels on gene targeting. Here we demonstrate that the transient knockdown of integral NHEJ proteins, Ku70 and Xrcc4, by RNAi in human HCT116 cells has a remarkable effect on gene targeting/random insertions ratios. A timely transfection of an HPRT-based targeting vector after RNAi treatment led to a 70% reduction in random integration events and a 33-fold increase in gene targeting at the HPRT locus. These findings bolster the role of NHEJ proteins in foreign DNA integration in vivo, and demonstrate that their transient depletion by RNAi is a viable approach to increase the frequency of gene targeting events. Understanding how foreign DNA integrates into a cell’s genome is important to advance strategies for biotechnology and genetic medicine.

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Acknowledgments

We thank Dr. Andrew Porter for kindly supplying the HPRT vector and Dr. Wolf D. Heyer for his assistance and valuable discussions during the experiments and for reading the manuscript. This study was supported by the UC Discovery Grant bio03-10412 with Invitrogen Corp. Luciana R. Bertolini was supported by a doctoral fellowship from CAPES/Brazil.

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Authors and Affiliations

  1. Department of Animal Science, University of California, Davis, CA, USA

    Luciana R. Bertolini, Marcelo Bertolini, Elizabeth A. Maga & James D. Murray

  2. Biogrammatics, Inc., Carlsbad, CA, USA

    Knut R. Madden

  3. Department of Population Health and Reproduction, University of California, Davis, CA, USA

    James D. Murray

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  1. Luciana R. Bertolini
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  2. Marcelo Bertolini
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  3. Elizabeth A. Maga
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  5. James D. Murray
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Correspondence to James D. Murray.

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Bertolini, L.R., Bertolini, M., Maga, E.A. et al. Increased Gene Targeting in Ku70 and Xrcc4 Transiently Deficient Human Somatic Cells. Mol Biotechnol 41, 106–114 (2009). https://doi.org/10.1007/s12033-008-9098-8

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