In vivo DNA double-strand breaks enhance gene targeting in cultured silkworm cells.
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).
Mon
Hiroaki
H
Laboratory of Silkworm Science, Faculty of Agriculture, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
Kusakabe
Takahiro
T
Lee
Jae Man
JM
Kawaguchi
Yutaka
Y
Koga
Katsumi
K
D000818:Animals; D012831:Bombyx / Q000166:cytology* / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D019457:Chromosome Breakage / Q000235:genetics*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018390:Gene Targeting / Q000295:instrumentation / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D049409:Luciferases, Firefly / Q000032:analysis / Q000235:genetics; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051135:Rad51 Recombinase; D012680:Sensitivity and Specificity
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D049409:Luciferases, Firefly; D011065:Poly(ADP-ribose) Polymerases; D051135:Rad51 Recombinase