DNA vector-based RNA interference in cell lines derived from Bombyx mori.
Gene-knockdown technology using RNA interference (RNAi) is widely used to characterize gene functions in many organisms. In this study, we analyzed the conditions for employing DNA vector-based RNAi in silkworm cell lines using long-hairpin RNA-expressing plasmid DNA. We found that NIAS-Bm-oyanagi2 was the most effective cell line for RNAi. Expression of long-hairpin RNA containing an approximately 500 base-pair stem region suppressed expression of a reporter target gene by more than 99% in this cell line. Furthermore, the loop sequence of hairpin RNA was not as important to RNAi efficiency as previously observed in Drosophila melanogaster. DNA vector-based RNAi also induced significant suppression of endogenous clathrin in NIAS-Bm-oyanagi2. Luciferase activity from recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) containing luciferase in the clathrin-knockdown cells was significantly less than in the control cells, suggesting that clathrin is indispensable for the entry of BmNPV into silkworm cell lines.
Fujita
Kosuke
K
Innate Immunity Research Unit, National Institute of Agrobiological Sciences, Ibaraki 305-8634, Japan.
Sagisaka
Aki
A
Tomimoto
Kazuya
K
Ishibashi
Jun
J
Imanishi
Shigeo
S
Yamakawa
Minoru
M
Tanaka
Hiromitsu
H
D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000166:cytology / Q000235:genetics*; D002460:Cell Line; D002966:Clathrin / Q000235:genetics; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D034622:RNA Interference; D014162:Transfection
D002966:Clathrin; D017931:DNA Primers; D004247:DNA