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• W2079397645 abstract "Gene targeting has two important applications. One is the inactivation of genes (“knockouts”), and the second is the correction of a mutated allele back to wild-type (“gene therapy”). Central to these processes is the efficient introduction of the targeting DNA into the cells of interest. In humans, this targeting is often accomplished through the use of recombinant adeno-associated virus (rAAV). rAAV is presumed to use a pathway of DNA double-strand break (DSB) repair termed homologous recombination (HR) to mediate correct targeting; however, the specifics of this mechanism remain unknown. In this work, we attempted to generate Ku70 -null human somatic cells by using a rAAV-based gene knockout strategy. Ku70 is the heterodimeric partner of Ku86, and together they constitute an end-binding activity that is required for a pathway [nonhomologous end joining (NHEJ)] of DSB repair that is believed to compete with HR. Our data demonstrated that Ku70 is an essential gene in human somatic cells. More importantly, however, in Ku70 +/− cells, the frequency of gene targeting was 5- to 10-fold higher than in wild-type cells. RNA interference and short-hairpinned RNA strategies to deplete Ku70 phenocopied these results in wild-type cells and greatly accentuated them in Ku70 +/− cell lines. Thus, Ku70 protein levels significantly influenced the frequency of rAAV-mediated gene targeting in human somatic cells. Our data suggest that gene-targeting frequencies can be significantly improved in human cells by impairing the NHEJ pathway, and we propose that Ku70 depletion can be used to facilitate both knockout and gene therapy approaches." @default.
• W2079397645 created "2016-06-24" @default.
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• W2079397645 date "2008-06-24" @default.
• W2079397645 modified "2023-09-27" @default.
• W2079397645 title "<i>Ku70</i> , an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells" @default.
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• W2079397645 doi "https://doi.org/10.1073/pnas.0712060105" @default.
• W2079397645 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2438404" @default.
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