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ZFIN ID: ZDB-PUB-080218-19
The Cytomegalovirus Promoter-Driven Short Hairpin RNA Constructs Mediate Effective RNA Interference in Zebrafish In Vivo
Su, J., Zhu, Z., Wang, Y., Xiong, F., and Zou, J.
Date: 2008
Source: Marine biotechnology (New York, N.Y.)   10(3): 262-269 (Journal)
Registered Authors: Su, Jianguo, Xiong, Feng
Keywords: CMV promoter, EGFP, No tail, RNAi, shRNA, Zebrafish
MeSH Terms:
  • Animals
  • Base Sequence
  • Cytomegalovirus/genetics*
  • Fetal Proteins
  • Genetic Vectors
  • Green Fluorescent Proteins/biosynthesis
  • Green Fluorescent Proteins/genetics
  • Promoter Regions, Genetic/genetics*
  • RNA Interference*
  • RNA, Small Interfering/genetics*
  • Sequence Alignment
  • T-Box Domain Proteins/biosynthesis
  • T-Box Domain Proteins/genetics
  • Zebrafish/genetics*
  • Zebrafish Proteins/biosynthesis
  • Zebrafish Proteins/genetics
PubMed: 18214611 Full text @ Mar. Biotechnol.
The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.