PUBLICATION
The Cytomegalovirus Promoter-Driven Short Hairpin RNA Constructs Mediate Effective RNA Interference in Zebrafish In Vivo
- Authors
- Su, J., Zhu, Z., Wang, Y., Xiong, F., and Zou, J.
- ID
- ZDB-PUB-080218-19
- 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
-
- RNA Interference*
- T-Box Domain Proteins/biosynthesis
- T-Box Domain Proteins/genetics
- Zebrafish Proteins/biosynthesis
- Zebrafish Proteins/genetics
- Sequence Alignment
- Animals
- Fetal Proteins
- Cytomegalovirus/genetics*
- Zebrafish/genetics*
- Promoter Regions, Genetic/genetics*
- Genetic Vectors
- RNA, Small Interfering/genetics*
- Green Fluorescent Proteins/biosynthesis
- Green Fluorescent Proteins/genetics
- Base Sequence
- PubMed
- 18214611 Full text @ Mar. Biotechnol.
Citation
Su, J., Zhu, Z., Wang, Y., Xiong, F., and Zou, J. (2008) The Cytomegalovirus Promoter-Driven Short Hairpin RNA Constructs Mediate Effective RNA Interference in Zebrafish In Vivo. Marine biotechnology (New York, N.Y.). 10(3):262-269.
Abstract
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping