Document Detail


Targeted chromosomal gene modification in human cells by single-stranded oligodeoxynucleotides in the presence of a DNA double-strand break.
MedLine Citation:
PMID:  16904944     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A DNA double-strand break (DSB) cannot be tolerated by a cell and is dealt with by several pathways. Here, it was hypothesized that DSB induction close to a targeted mutation in the genome of a mammalian cell might attract oligodeoxynucleotide (ODN)-directed gene repair. A HEK-293-derived cell line had been engineered harboring a single target locus with open reading frames encoding the living-cell reporter proteins LacZ and EGFP, the latter translationally decoupled by a DNA spacer with a unique I-SceI recognition site for defined DSB induction. To enable expression of a fluorescent LacZ-EGFP fusion protein, single-stranded (ss) ODNs (80 or 96 nucleotides long) spanning the DSB were designed to fuse both reading frames by altering a few base-pair positions, deleting 59 bp or introducing a 10-bp fragment. The ssODNs alone generated few EGFP-positive cells. With I-SceI transiently expressed, more than 0.3% of cells revealed EGFP expression 7 days after transfection, with up to 96% of the loci faithfully corrected, depending on the ssODN used. During these correction events, the ssODN did not become physically incorporated into the chromosome, but served only as information template. Unwanted insertional mutagenesis also occurred. Both observations have important implications for gene therapy.
Authors:
Frank Radecke; Ingrid Peter; Sarah Radecke; Katharina Gellhaus; Klaus Schwarz; Toni Cathomen
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2006-08-14
Journal Detail:
Title:  Molecular therapy : the journal of the American Society of Gene Therapy     Volume:  14     ISSN:  1525-0016     ISO Abbreviation:  Mol. Ther.     Publication Date:  2006 Dec 
Date Detail:
Created Date:  2006-11-22     Completed Date:  2007-01-30     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  100890581     Medline TA:  Mol Ther     Country:  United States    
Other Details:
Languages:  eng     Pagination:  798-808     Citation Subset:  IM    
Affiliation:
Department of Transfusion Medicine, University Hospital Ulm, and Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, 89081 Ulm, Germany.
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MeSH Terms
Descriptor/Qualifier:
Base Sequence
Cell Line
DNA Breaks, Double-Stranded*
DNA Repair / genetics*
DNA, Single-Stranded / genetics
Green Fluorescent Proteins / genetics
Humans
Lac Operon / genetics
Models, Genetic
Molecular Sequence Data
Oligodeoxyribonucleotides / genetics*
Transfection
Chemical
Reg. No./Substance:
0/DNA, Single-Stranded; 0/Oligodeoxyribonucleotides; 0/enhanced green fluorescent protein; 147336-22-9/Green Fluorescent Proteins

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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