PUBLICATION

Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

Authors
Wang, N., Sun, Y.H., Liu, J., Wu, G., Su, J.G., Wang, Y.P., and Zhu, Z.Y.
ID
ZDB-PUB-070726-8
Date
2007
Source
Journal of Biomedical Science   14(6): 767-776 (Journal)
Registered Authors
Liu, Jing, Sun, Yonghua
Keywords
short hairpin RNA, T7 RNA polymerase, T7 promoter, zebrafish, fluorescence real time RT-PCR, whole mount in situ hybridization
MeSH Terms
  • Transcription, Genetic
  • Green Fluorescent Proteins/antagonists & inhibitors
  • Green Fluorescent Proteins/genetics*
  • Green Fluorescent Proteins/metabolism
  • Fetal Proteins
  • Zebrafish/embryology*
  • Zebrafish/genetics
  • Animals
  • Plasmids/genetics*
  • Plasmids/metabolism
  • Gene Expression Regulation, Developmental
  • Embryo, Nonmammalian/metabolism
  • T-Box Domain Proteins/antagonists & inhibitors
  • T-Box Domain Proteins/genetics*
  • T-Box Domain Proteins/metabolism
  • RNA Interference*
  • RNA, Untranslated/genetics*
  • RNA, Untranslated/metabolism
  • Zebrafish Proteins/antagonists & inhibitors
  • Zebrafish Proteins/genetics*
  • Zebrafish Proteins/metabolism
(all 21)
PubMed
17624603 Full text @ J. Biomed. Sci.
Abstract
A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 microg/microl. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.
Genes / Markers
Figures
No images available
Expression
Phenotype
No data available
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
zf79TgTransgenic Insertion
    1 - 1 of 1
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    Human Disease / Model
    No data available
    Sequence Targeting Reagents
    No data available
    Fish
    Fish
    zf79Tg (AB)
    1 - 1 of 1
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    Antibodies
    No data available
    Orthology
    No data available
    Engineered Foreign Genes
    Marker Marker Type Name
    EGFPEFGEGFP
    1 - 1 of 1
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    Mapping
    No data available
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    Citation
    Wang, N., Sun, Y.H., Liu, J., Wu, G., Su, J.G., Wang, Y.P., and Zhu, Z.Y. (2007) Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system. Journal of Biomedical Science. 14(6):767-776.