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Targeted gene knockout mediated by triple helix forming oligonucleotides
A Majumdar1, A Khorlin, N Dyatkina
1National Institute on Aging, NIH, Baltimore, Maryland 21224-6825, USA.
Nature Genetics
|October 15, 1998
Summary
Modified triple helix forming oligonucleotides (TFOs) successfully targeted the Hprt gene in viable cells. This demonstrates TFOs
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Triple helix forming oligonucleotides (TFOs) bind specific DNA sequences.
- TFOs show potential for targeted genomic applications.
- Previous studies faced challenges with chromatin structure and cellular targeting.
Purpose of the Study:
- To demonstrate chromosome targeting by TFOs in viable cells.
- To investigate the efficacy of psoralen-linked TFOs at the Hprt gene.
- To assess the impact of TFOs on chromatin structure dynamics.
Main Methods:
- Modified TFOs linked to psoralen were designed.
- TFOs were introduced into viable cells targeting the Hprt gene.
- Photoactivation of psoralen induced DNA crosslinking.
- Mutation analysis was performed on recovered cellular clones.
Main Results:
- Stable Hprt-deficient clones were recovered after TFO treatment and photoactivation.
- 85% of analyzed clones (282 total) exhibited mutations within the TFO target region.
- Predominant mutations included deletions and some insertions.
Conclusions:
- Constructed TFOs can effectively target chromosomal DNA in living cells.
- The study suggests greater dynamism in target chromatin structure than previously predicted.
- This work validates TFOs as tools for site-specific genomic modification in cellular contexts.