Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting.
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.
Iiizumi
Susumu
S
International Graduate School of Arts and Sciences and Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan.
Kurosawa
Aya
A
So
Sairei
S
Ishii
Yasuyuki
Y
Chikaraishi
Yuichi
Y
Ishii
Ayako
A
Koyama
Hideki
H
Adachi
Noritaka
N
D000818:Animals; D045744:Cell Line, Tumor; D002645:Chickens / Q000235:genetics; D004247:DNA / Q000378:metabolism; D011088:DNA Ligases / Q000235:genetics; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D011995:Recombination, Genetic
D004247:DNA; D011088:DNA Ligases