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Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells
13:10

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

Published on: September 8, 2010

Double-stranded break can be repaired by single-stranded oligonucleotides via the ATM/ATR pathway in mammalian cells

Zai Wang1, Zhong-Jun Zhou, De-Pei Liu

  • 1Department of Biochemistry, The University of Hong Kong, Hong Kong SAR, People's Republic of China.

Oligonucleotides
|March 7, 2008
PubMed

Insights

This study introduces a new method for gene repair using single-stranded oligonucleotides (SSOs) and double-stranded break (DSB) induction, enabling viable cell division after gene modification.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Single-stranded oligonucleotide (SSO)-mediated gene modification enables site-specific gene repair in mammalian cells.
  • However, a significant challenge is the G2/M arrest and lack of cell division in corrected cells, hindering clinical applications.

Purpose of the Study:

  • To develop an efficient SSO-mediated DNA repair system that overcomes the limitations of previous methods.
  • To investigate the mechanism and optimize SSO-mediated gene repair using double-stranded break (DSB) induction.

Main Methods:

  • Generated a mutant enhanced green fluorescent protein (EGFP) reporter gene with varying insertion lengths (24 bp to 1.6 kb) in mammalian cell lines.
  • Utilized SSOs to delete insertion fragments near an induced DSB.
  • Investigated the role of the ATM/ATR pathway in the repair process.

Main Results:

  • Successfully deleted insertion fragments using SSOs upon DSB induction.
  • Demonstrated that the repair process is dependent on the ATM/ATR signaling pathway.
  • Achieved viable repaired cell clones, overcoming previous limitations of cell cycle arrest.

Conclusions:

  • The developed SSO-mediated DNA repair system based on DSB induction is efficient and yields viable cells.
  • This approach offers a promising strategy for site-specific gene repair and future therapeutic applications.
  • Further studies investigated factors influencing gene repair frequency, including deletion length, SSO characteristics, and modifications.

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