Physical incorporation of a single-stranded oligodeoxynucleotide during targeted repair of a human chromosomal locus

• Published in: The journal of gene medicine 2006-02, Vol.8 (2), p.217-228
• Main Authors: Radecke, Sarah, Radecke, Frank, Peter, Ingrid, Schwarz, Klaus
• Format: Article
• Language: English
• Subjects: Biotin - metabolism; Cell Line; Codon, Nonsense; dideoxy terminus; DNA; DNA Repair - physiology; Gene Targeting; Gene therapy; Genes, Reporter; Genetic Therapy; Humans; Molecular Probes; Okazaki fragment; Oligodeoxyribonucleotides - metabolism; oligonucleotide incorporation; oligonucleotide-mediated gene correction
• ISSN: 1099-498X; 1521-2254
• DOI: 10.1002/jgm.828

Background Targeted gene repair is an attractive method to correct point‐mutated genes at their natural chromosomal sites, but it is still rather inefficient. As revealed by earlier studies, successful gene correction requires a productive interaction of the repair molecule with the target locus. The work here set out to investigate whether DNA repair, e.g., mismatch repair, or a direct incorporation of the correction molecule follows as the step upon the initial interaction. Methods Single‐stranded 21mer oligodeoxynucleotides (ODNs) of sense orientation were directed towards point‐mutated enhanced green fluorescence protein transgene loci in HEK‐293‐derived cell clones. First gene repair assays compared ODNs carrying the canonical termini 5′‐phosphate and 3′‐OH with their respective variants harbouring non‐canonical termini (5′‐OH, 3′‐H). Second, a protocol was established to allow efficient recovery of integrated short biotin‐labelled ODNs from the genomes of gene‐corrected cells using streptavidin‐coated beads in order to test directly whether transfected ODNs become bona fide parts of the target locus DNA. Results Oligodeoxynucleotides with canonical termini were about 34‐fold more efficient than their counterparts carrying non‐canonical termini in a phosphorothioate‐modified backbone. Furthermore, biotinylated fragments were successfully recovered from genomic DNAs of gene‐corrected cells. Conclusions The experiment showed that ODNs are incorporated into a mammalian genome. This unravels one early repair step and also sets an unexpected example of genome dynamics possibly relevant to other ODN‐based cell techniques. Copyright © 2005 John Wiley & Sons, Ltd.