Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2005-03

AUTHORS

P A Olsen, M Randol, S Krauss

ABSTRACT

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. More... »

PAGES

3302454

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/sj.gt.3302454

DOI

http://dx.doi.org/10.1038/sj.gt.3302454

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1051370883

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/15674399


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