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

Marine Biotechnology
Jianguo SuJun Zou

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...Continue Reading

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Citations

Jun 13, 2013·Developmental and Comparative Immunology·Youliang RaoQingmei Li
Apr 16, 2010·Zebrafish·Ilya A Shestopalov, James K Chen
Apr 29, 2011·Briefings in Functional Genomics·Amanda Kelly, Adam F Hurlstone
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Oct 2, 2012·Journal of Genetics and Genomics = Yi Chuan Xue Bao·Peng HuangBo Zhang
May 3, 2008·Biochemical and Biophysical Research Communications·Kosuke Zenke, Ki Hong Kim
Aug 3, 2013·Marine Biotechnology·Sajad AhangerRakesh K Mishra
Nov 14, 2008·European Spine Journal : Official Publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society·Kotaro NishidaMinoru Doita
Jan 7, 2020·Journal of Neurogenetics·Sergey V CheresizAllan V Kalueff

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