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

Iiizumi, Susumu, Kurosawa, Aya, So, Sairei, Ishii, Yasuyuki, Chikaraishi, Yuichi, Ishii, Ayako, Koyama, Hideki, Adachi, Noritaka
Nucleic acids research 2008 v.36 no.19 pp. 6333-6342
DNA, chickens, gene targeting, genome, homologous recombination, humans
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.