In vivo DNA double-strand breaks enhance gene targeting in cultured silkworm cells

Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology
Hiroaki MonKatsumi Koga

Abstract

Alteration of genomic information through homologous recombination (HR) is a powerful tool for reverse genetics in bacteria, yeast, and mice. The low frequency of HR is, however, a major obstacle to achieve efficient gene targeting. In this study, we have developed an assay system for investigating the frequency of gene targeting in cultured silkworm cells using a firefly luciferase gene as a reporter. The introduction of a DNA double-strand break (DSB) either in the chromosomal target locus or in the targeting construct drastically increased the frequency of gene targeting. Interestingly, the inhibition of poly(ADP-ribose) polymerase (PARP), a protein known to play an important role in overall suppression of the HR pathway, stimulated the targeting efficiency, whereas the overexpression of two silkworm RecA homologs, BmRad51 and BmDmc1, had no effect. The presently devised assay system may serve as a useful tool to improve the gene targeting efficiency in the silkworm (Bombyx mori).

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Citations

Aug 16, 2011·Molecular Genetics and Genomics : MGG·Hiroaki MonTakahiro Kusakabe
Feb 16, 2006·Nucleic Acids Research·Haruna TsukiokaTakahiro Kusakabe
Apr 30, 2014·Journal of Insect Science·Hiroaki MonTakahiro Kusakabe
Jun 23, 2018·Physical Chemistry Chemical Physics : PCCP·Grzegorz ZajacMalgorzata Baranska
Mar 10, 2015·Journal of Food Science and Technology·Pedro Cerezal MezquitaClaudia P Ortíz Hinojosa

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