The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo

J Su, Z Zhu, Y Wang, F Xiong, J Zou

Research output: Contribution to journalArticle

21 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)262-269
Number of pages8
JournalMarine Biotechnology
Volume10
Issue number3
Early online date24 Jan 2008
DOIs
Publication statusPublished - May 2008

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Cytomegalovirus
Zebrafish
small interfering RNA
RNA Interference
RNA interference
Danio rerio
green fluorescent protein
Small Interfering RNA
RNA
promoter regions
protein
plasmid
plasmids
Plasmids
gene expression
Tail
tail
Gene Expression
polymerase chain reaction
Reverse Transcription

Cite this

The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo. / Su, J; Zhu, Z; Wang, Y; Xiong, F; Zou, J.

In: Marine Biotechnology, Vol. 10, No. 3, 05.2008, p. 262-269.

Research output: Contribution to journalArticle

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