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Nucleic Acids Research Advance Access published online on October 3, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn649
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Genome integrity, repair and replication

Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting

Susumu Iiizumi1,2, Aya Kurosawa1, Sairei So2, Yasuyuki Ishii2, Yuichi Chikaraishi2, Ayako Ishii1, Hideki Koyama2 and Noritaka Adachi1,2,*

1International Graduate School of Arts and Sciences and 2Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan

*To whom correspondence should be addressed. Tel/Fax: +81 45 787 2228; Email: nadachi{at}yokohama-cu.ac.jp

Correspondence may also be addressed to Hideki Koyama. Email: koyama{at}yokohama-cu.ac.jp

Received August 12, 2008. Revised September 18, 2008. Accepted September 18, 2008.

In higher animal cells, the principal limitation of gene-targeting technology is the extremely low efficiency of targeted integration, which occurs three to four orders of magnitude less frequently than random integration. Assuming that random integration mechanistically involves non-homologous end-joining (NHEJ), inactivation of this pathway should reduce random integration and may enhance gene targeting. To test this possibility, we examined the frequencies of random and targeted integration in NHEJ-deficient chicken DT40 and human Nalm-6 cell lines. As expected, loss of NHEJ resulted in drastically reduced random integration in DT40 cells. Unexpectedly, however, this was not the case for Nalm-6 cells, indicating that NHEJ is not the sole mechanism of random integration. Nevertheless, we present evidence that NHEJ inactivation can lead to enhanced gene targeting through a reduction of random integration and/or an increase in targeted integration by homologous recombination. Most intriguingly, our results show that, in the absence of functional NHEJ, random integration of targeting vectors occurs more frequently than non-targeting vectors (harboring no or little homology to the host genome), implying that suppression of NHEJ-independent random integration events is needed to greatly enhance gene targeting in animal cells.


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