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

Hiroaki Mon, Takahiro Kusakabe, Man Lee, Yutaka Kawaguchi, Katsumi Koga

Research output: Contribution to journalArticle

10 Citations (Scopus)

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).

Original languageEnglish
Pages (from-to)99-106
Number of pages8
JournalComparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
Volume139
Issue number1
DOIs
Publication statusPublished - Sep 1 2004

Fingerprint

Bombyx
Gene Targeting
Double-Stranded DNA Breaks
Cultured Cells
Homologous Recombination
Genes
DNA
Assays
Firefly Luciferases
Reverse Genetics
Poly(ADP-ribose) Polymerases
Yeasts
Yeast
Bacteria
Proteins

All Science Journal Classification (ASJC) codes

  • Physiology
  • Biochemistry
  • Molecular Biology

Cite this

In vivo DNA double-strand breaks enhance gene targeting in cultured silkworm cells. / Mon, Hiroaki; Kusakabe, Takahiro; Lee, Man; Kawaguchi, Yutaka; Koga, Katsumi.

In: Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology, Vol. 139, No. 1, 01.09.2004, p. 99-106.

Research output: Contribution to journalArticle

@article{57d7730dfd10408598452c89813a48b4,
title = "In vivo DNA double-strand breaks enhance gene targeting in cultured silkworm cells",
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).",
author = "Hiroaki Mon and Takahiro Kusakabe and Man Lee and Yutaka Kawaguchi and Katsumi Koga",
year = "2004",
month = "9",
day = "1",
doi = "10.1016/j.cbpc.2004.06.013",
language = "English",
volume = "139",
pages = "99--106",
journal = "Comparative biochemistry and physiology. B, Comparative biochemistry",
issn = "1096-4959",
publisher = "Elsevier Inc.",
number = "1",

}

TY - JOUR

T1 - In vivo DNA double-strand breaks enhance gene targeting in cultured silkworm cells

AU - Mon, Hiroaki

AU - Kusakabe, Takahiro

AU - Lee, Man

AU - Kawaguchi, Yutaka

AU - Koga, Katsumi

PY - 2004/9/1

Y1 - 2004/9/1

N2 - 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).

AB - 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).

UR - http://www.scopus.com/inward/record.url?scp=4444321506&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=4444321506&partnerID=8YFLogxK

U2 - 10.1016/j.cbpc.2004.06.013

DO - 10.1016/j.cbpc.2004.06.013

M3 - Article

C2 - 15364292

AN - SCOPUS:4444321506

VL - 139

SP - 99

EP - 106

JO - Comparative biochemistry and physiology. B, Comparative biochemistry

JF - Comparative biochemistry and physiology. B, Comparative biochemistry

SN - 1096-4959

IS - 1

ER -