title: Properties of gene knockdown system by vector-based siRNA in zebrafish.
abstract: RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector-based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue-specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene-silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6-shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6-shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.
authors.affiliation: Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.|The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Minori
authors.initials: M
authors.lastname: Shinya
authors.affiliation: Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Kayo
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Aki
authors.initials: A
authors.lastname: Masuda
authors.affiliation: Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Tokumoto
authors.affiliation: Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Ozaki
authors.affiliation: Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Saito
authors.affiliation: Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Kawasaki
authors.affiliation: Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Sado
authors.affiliation: Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.|The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, 411-8540, Japan.
authors.forename: Noriyoshi
authors.initials: N
authors.lastname: Sakai
mesh_terms: D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005260:Female; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015027:Zebrafish / Q000235:genetics*
chemical_list: D035683:MicroRNAs; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012321:DNA-Directed RNA Polymerases
keywords: RNAi; U6 snRNA promoter; shRNA; siRNA