PUBLICATION

Properties of gene knockdown system by vector-based siRNA in zebrafish

Authors
Shinya, M., Kobayashi, K., Masuda, A., Tokumoto, M., Ozaki, Y., Saito, K., Kawasaki, T., Sado, Y., and Sakai, N.
ID
ZDB-PUB-131113-9
Date
2013
Source
Development, growth & differentiation   55(9): 755-765 (Journal)
Registered Authors
Sado, Yukiko, Sakai, Noriyoshi, Shinya, Minori
Keywords
RNAi, shRNA, siRNA, U6 snRNA promoter
MeSH Terms
  • Animals
  • Base Sequence
  • Blotting, Northern
  • Cloning, Molecular
  • DNA-Directed RNA Polymerases/genetics
  • Embryo, Nonmammalian/metabolism
  • Female
  • Gene Knockdown Techniques/methods*
  • Genetic Vectors/genetics*
  • Green Fluorescent Proteins/genetics
  • Green Fluorescent Proteins/metabolism
  • Male
  • MicroRNAs/genetics*
  • Molecular Sequence Data
  • RNA Interference*
  • RNA, Small Interfering/genetics*
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Zebrafish/genetics*
PubMed
24117364 Full text @ Dev. Growth Diff.
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.

Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping