Impact of Non-homologous End-joining Deficiency on Random and Targeted DNA Integration: Implications for Gene Targeting

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2008 Oct 7

PMID 18835848

Citations 28

Authors

Susumu Iiizumi

Aya Kurosawa

Sairei So

Yasuyuki Ishii

Yuichi Chikaraishi

Ayako Ishii

Hideki Koyama

Noritaka Adachi

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

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Autopolyploidization affects transcript patterns and gene targeting frequencies in Physcomitrella.

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