Document Detail


Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells.
MedLine Citation:
PMID:  18562296     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Farjana J Fattah; Natalie F Lichter; Kazi R Fattah; Sehyun Oh; Eric A Hendrickson
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2008-06-18
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  105     ISSN:  1091-6490     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  2008 Jun 
Date Detail:
Created Date:  2008-06-25     Completed Date:  2008-08-19     Revised Date:  2013-06-05    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  8703-8     Citation Subset:  IM    
Affiliation:
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
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MeSH Terms
Descriptor/Qualifier:
Antigens, Nuclear / genetics*
Cell Line, Tumor
DNA-Binding Proteins / genetics*,  metabolism
Dependovirus / genetics*
Gene Targeting*
Gene Transfer Techniques
Genetic Vectors / metabolism
HCT116 Cells
Humans
Recombination, Genetic
Transfection
Grant Support
ID/Acronym/Agency:
GM 069576/GM/NIGMS NIH HHS; HL079559/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/Antigens, Nuclear; 0/DNA-Binding Proteins; 0/Ku autoantigen
Comments/Corrections

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