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Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system
Wang, Na; Sun, Yong-Hua; Liu, Jing; Wu, Gang; Su, Jian-Guo; Wang, Ya-Ping; Zhu, Zuo-Yan; Sun, YH, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, 7 Donghu S Rd, Wuhan 430072, Peoples R China
2007-11-01
Source PublicationJOURNAL OF BIOMEDICAL SCIENCE
ISSN1021-7770
Volume14Issue:6Pages:767-776
AbstractA short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.; A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.
SubtypeArticle
KeywordShort Hairpin Rna T7 Rna Polymerase T7 Promoter Zebrafish Fluorescence Real Time Rt-pcr Whole Mount In Situ Hybridization
DepartmentChinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China; Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China
Subject AreaMedicine ; Research & Experimental
DOI10.1007/s11373-007-9189-8
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
Indexed BySCI
Language英语
WOS Research AreaResearch & Experimental Medicine
WOS SubjectMedicine, Research & Experimental
WOS IDWOS:000250119200006
WOS KeywordDOUBLE-STRANDED-RNA ; SMALL INTERFERING RNA ; MAMMALIAN-CELLS ; GENE-EXPRESSION ; C-ELEGANS ; T7-RNA POLYMERASE ; CLONED CDNAS ; FLOOR PLATE ; SIRNA ; PROMOTER
Citation statistics
Cited Times:11[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.ihb.ac.cn/handle/152342/8396
Collection期刊论文
Corresponding AuthorSun, YH, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, 7 Donghu S Rd, Wuhan 430072, Peoples R China
Affiliation1.Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China
2.Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China
Recommended Citation
GB/T 7714
Wang, Na,Sun, Yong-Hua,Liu, Jing,et al. Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system[J]. JOURNAL OF BIOMEDICAL SCIENCE,2007,14(6):767-776.
APA Wang, Na.,Sun, Yong-Hua.,Liu, Jing.,Wu, Gang.,Su, Jian-Guo.,...&Sun, YH, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, 7 Donghu S Rd, Wuhan 430072, Peoples R China.(2007).Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system.JOURNAL OF BIOMEDICAL SCIENCE,14(6),767-776.
MLA Wang, Na,et al."Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system".JOURNAL OF BIOMEDICAL SCIENCE 14.6(2007):767-776.
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