Enhanced oligonucleotide-directed gene targeting in mammalian cells following treatment with DNA damaging agents

Experimental Cell Research
Luciana FerraraE B Kmiec

Abstract

Targeted gene repair, a form of oligonucleotide-directed mutagenesis, employs end-modified single-stranded DNA oligonucleotides to mediate single-base changes in chromosomal DNA. In this work, we use a specific 72-mer to direct the repair of a mutated eGFP gene stably integrated in the genome of DLD-1 cells. Corrected cells express eGFP that can be identified and quantitated by FACS. The repair of this mutant gene is dependent on the presence of a specifically designed oligonucleotide and the frequency with which the mutation is reversed is affected by the induction of DNA damage. We used hydroxyurea, VP16 (etoposide), and thymidine to modulate the rate of DNA replication through the stalling of the replication forks or the introduction of lesions. Addition of hydroxyurea or VP16 before the electroporation of the oligonucleotide, results in an accumulation of double-strand breaks (DSB) whose repair is facilitated by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The addition of thymidine results in DNA damage within replication forks, damage that is repaired through the process of homologous recombination. Our data suggest that gene repair activity is elevated when DNA damage induces or activates the ...Continue Reading

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Citations

Feb 8, 2005·Proceedings of the National Academy of Sciences of the United States of America·Xue-Song WuChih-Chuan Liang
Nov 8, 2006·Nucleic Acids Research·Michael S Y HuenJian-Dong Huang
Jan 30, 2007·BMC Molecular Biology·Timothy R Schwartz, Eric B Kmiec
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Jan 25, 2005·Oligonucleotides·Olga IgouchevaKyonggeun Yoon
Oct 6, 2005·Oligonucleotides·Miya D DruryEric B Kmiec
Jun 15, 2010·Human Molecular Genetics·Refik KayaliCarmen Bertoni
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May 26, 2009·Mutation Research·Melissa Bonner, Eric B Kmiec
Jun 15, 2007·DNA Repair·Luciana FerraraEric B Kmiec
Jan 22, 2014·Scientific Reports·Bryan StrouseEric B Kmiec
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May 1, 2021·International Journal of Molecular Sciences·Kevin Bloh, Natalia Rivera-Torres

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