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The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo
Su, Jianguo1,2; Zhu, Zuoyan1; Wang, Yaping1; Xiong, Feng1; Zou, Jun3; Zhu, ZY, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China
2008-05-01
Source PublicationMARINE BIOTECHNOLOGY
ISSN1436-2228
Volume10Issue:3Pages:262-269
AbstractThe 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.; 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.
SubtypeArticle
KeywordCmv Promoter Egfp No Tail Rnai Shrna Zebrafish
Department[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
Subject AreaBiotechnology & Applied Microbiology ; Marine & Freshwater Biology
DOI10.1007/s10126-007-9059-4
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
Indexed BySCI
Language英语
WOS Research AreaBiotechnology & Applied Microbiology ; Marine & Freshwater Biology
WOS SubjectBiotechnology & Applied Microbiology ; Marine & Freshwater Biology
WOS IDWOS:000254905500006
WOS KeywordDOUBLE-STRANDED-RNA ; MAMMALIAN-CELLS ; CAENORHABDITIS-ELEGANS ; EXPRESSION VECTORS ; POLYMERASE-III ; GENE ; INJECTION ; EMBRYOS ; SYSTEM ; INHIBITION
Citation statistics
Cited Times:21[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.ihb.ac.cn/handle/152342/8176
Collection期刊论文
Corresponding AuthorZhu, ZY, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China
Affiliation1.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
GB/T 7714
Su, Jianguo,Zhu, Zuoyan,Wang, Yaping,et al. The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo[J]. MARINE BIOTECHNOLOGY,2008,10(3):262-269.
APA Su, Jianguo,Zhu, Zuoyan,Wang, Yaping,Xiong, Feng,Zou, Jun,&Zhu, ZY, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China.(2008).The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo.MARINE BIOTECHNOLOGY,10(3),262-269.
MLA Su, Jianguo,et al."The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo".MARINE BIOTECHNOLOGY 10.3(2008):262-269.
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