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学科主题: Medicine ; Research & Experimental
题名: Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system
作者: Wang, Na; Sun, Yong-Hua; Liu, Jing; Wu, Gang; Su, Jian-Guo; Wang, Ya-Ping; Zhu, Zuo-Yan
通讯作者: Sun, YH, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, 7 Donghu S Rd, Wuhan 430072, Peoples R China
关键词: short hairpin RNA ; T7 RNA polymerase ; T7 promoter ; zebrafish ; fluorescence real time RT-PCR ; whole mount in situ hybridization
刊名: JOURNAL OF BIOMEDICAL SCIENCE
发表日期: 2007-11-01
DOI: 10.1007/s11373-007-9189-8
卷: 14, 期:6, 页:767-776
收录类别: SCI
文章类型: Article
部门归属: Chinese 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
WOS标题词: Science & Technology ; Life Sciences & Biomedicine
类目[WOS]: Medicine, Research & Experimental
研究领域[WOS]: Research & Experimental Medicine
摘要: 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.
英文摘要: 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.
关键词[WOS]: DOUBLE-STRANDED-RNA ; SMALL INTERFERING RNA ; MAMMALIAN-CELLS ; GENE-EXPRESSION ; C-ELEGANS ; T7-RNA POLYMERASE ; CLONED CDNAS ; FLOOR PLATE ; SIRNA ; PROMOTER
语种: 英语
WOS记录号: WOS:000250119200006
ISSN号: 1021-7770
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/152342/8396
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.Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China

Recommended Citation:
Wang, Na; Sun, Yong-Hua; Liu, Jing; Wu, Gang; Su, Jian-Guo; Wang, Ya-Ping; Zhu, Zuo-Yan.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,2007,14(6):767-776
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