Document Detail

Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides.
MedLine Citation:
PMID:  15674399     Owner:  NLM     Status:  MEDLINE    
Oligonucleotide-based sequence alteration in living cells is a substantial methodological challenge in gene therapy. Here, we demonstrate that using corrective single-stranded oligonucleotides (ssODN), high and reproducible sequence correction rates can be obtained. CHO cell lines with chromosomally integrated multiple copy EGFP reporter genes routinely show rates of 4.5% targeted sequence correction after transfection with ssODN. We demonstrate that the cell cycle influences the rates of targeted sequence correction in vivo, with a peak in the early S phase during ssODN exposure. After cell division, the altered genomic sequence is predominantly passed to one daughter cell, indicating that targeted sequence alteration occurs after the replication fork has passed over the targeted site. Although high initial correction rates can be obtained by this method, we show that a majority of the corrected cells arrest in the G2/M cell cycle phase, although 1-2% of the corrected cells form viable colonies. The G2/M arrest observed after targeted sequence correction can be partially released by caffeine, pentoxifylline or Go6976 exposure. Despite substantial remaining challenges, targeted sequence alteration based on ssODN increasingly promises to become a powerful tool for functional gene alterations.
P A Olsen; M Randol; S Krauss
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Gene therapy     Volume:  12     ISSN:  0969-7128     ISO Abbreviation:  Gene Ther.     Publication Date:  2005 Mar 
Date Detail:
Created Date:  2005-03-09     Completed Date:  2005-06-09     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  9421525     Medline TA:  Gene Ther     Country:  England    
Other Details:
Languages:  eng     Pagination:  546-51     Citation Subset:  IM    
Department for Cellular and Genetic Therapy, Institute for Microbiology, Rikshospitalet, Forskningsparken, Oslo, Norway.
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MeSH Terms
CHO Cells
Cell Division
DNA Repair*
DNA, Single-Stranded / administration & dosage*
Flow Cytometry
Gene Therapy / methods*
Green Fluorescent Proteins / genetics
S Phase
Transfection / methods*
Reg. No./Substance:
0/DNA, Single-Stranded; 147336-22-9/Green Fluorescent Proteins

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

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