shRNA expression plasmids generated by a novel method efficiently induce gene-specific knockdown in a silkworm cell line.
RNAi knockdown by using shRNA expression plasmids is widely used to determine the function of individual genes in mammals. Here we developed a simple method to create an IR DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded IR DNA with short hairpin structure and Bst DNA polymerase. Furthermore, we demonstrated that the shRNA expression plasmids constructed by our method effectively induced target-specific RNAi in the silkworm cell line. We also found that sequence preference in the silkworm cell line was much lower than in mammalian cells and shRNA-induced RNAi was influenced by the length of the stem region.
Tanaka
Hiromitsu
H
Innate Immunity Research Unit, National Institute of Agrobiological Sciences, Ibaraki, 305-8634, Japan. htanaka1@affrc.go.jp
Fujita
Kosuke
K
Sagisaka
Aki
A
Tomimoto
Kazuya
K
Imanishi
Shigeo
S
Yamakawa
Minoru
M
D000818:Animals; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D055785:Gene Knockdown Techniques / Q000379:methods*; D055029:Inverted Repeat Sequences / Q000502:physiology; D049410:Luciferases, Renilla / Q000378:metabolism; D049408:Luminescent Agents / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D034622:RNA Interference / Q000502:physiology*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics
D049408:Luminescent Agents; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D049410:Luciferases, Renilla