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学科主题: Biotechnology & Applied Microbiology; Marine & Freshwater Biology
题名: The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo
作者: Su, Jianguo1, 2; Zhu, Zuoyan1; Wang, Yaping1; Xiong, Feng1; Zou, Jun3
通讯作者: Zhu, ZY, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China
关键词: CMV promoter ; EGFP ; no tail ; RNAi ; shRNA ; zebrafish
刊名: MARINE BIOTECHNOLOGY
发表日期: 2008-05-01
DOI: 10.1007/s10126-007-9059-4
卷: 10, 期:3, 页:262-269
收录类别: SCI
文章类型: Article
部门归属: [Su, Jianguo; Zhu, Zuoyan; Wang, Yaping; Xiong, Feng] Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China; [Su, Jianguo] NW A&F Univ, Coll Anim Sci & Technol, Dept Aquaculture, Yangling 712100, Peoples R China; [Zou, Jun] Univ Aberdeen, Sch Biol Sci, Aberdeen AB24 2TZ, Scotland
WOS标题词: Science & Technology ; Life Sciences & Biomedicine
类目[WOS]: Biotechnology & Applied Microbiology ; Marine & Freshwater Biology
研究领域[WOS]: Biotechnology & Applied Microbiology ; Marine & Freshwater Biology
摘要: 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.
英文摘要: 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.
关键词[WOS]: DOUBLE-STRANDED-RNA ; MAMMALIAN-CELLS ; CAENORHABDITIS-ELEGANS ; EXPRESSION VECTORS ; POLYMERASE-III ; GENE ; INJECTION ; EMBRYOS ; SYSTEM ; INHIBITION
语种: 英语
WOS记录号: WOS:000254905500006
ISSN号: 1436-2228
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/152342/8176
Appears in Collections:中科院水生所知识产出(2009年前)_期刊论文

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作者单位: 1.Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China
2.NW A&F Univ, Coll Anim Sci & Technol, Dept Aquaculture, Yangling 712100, Peoples R China
3.Univ Aberdeen, Sch Biol Sci, Aberdeen AB24 2TZ, Scotland

Recommended Citation:
Su, Jianguo; Zhu, Zuoyan; Wang, Yaping; Xiong, Feng; Zou, Jun.The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo,MARINE BIOTECHNOLOGY,2008,10(3):262-269
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