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.
International Graduate School of Arts and Sciences and Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan.
Susumu
S
Iiizumi
Aya
A
Kurosawa
Sairei
S
So
Yasuyuki
Y
Ishii
Yuichi
Y
Chikaraishi
Ayako
A
Ishii
Hideki
H
Koyama
Noritaka
N
Adachi
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