Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

CORC > 水生生物研究所 > 中科院水生所知识产出（2009年前） > 期刊论文

	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
刊名	JOURNAL OF BIOMEDICAL SCIENCE
	2007-11-01
卷号	14 期号:6 页码:767-776
关键词	short hairpin RNA T7 RNA polymerase T7 promoter zebrafish fluorescence real time RT-PCR whole mount in situ hybridization
ISSN号	1021-7770
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
中文摘要	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.
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.
学科主题	Medicine ; Research & Experimental
WOS标题词	Science & Technology ; Life Sciences & Biomedicine
类目[WOS]	Medicine, Research & Experimental
研究领域[WOS]	Research & Experimental Medicine
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
收录类别	SCI
语种	英语
WOS记录号	WOS:000250119200006
公开日期	2010-10-13
内容类型	期刊论文
源URL	[http://ir.ihb.ac.cn/handle/152342/8396]
专题	水生生物研究所_中科院水生所知识产出（2009年前）_期刊论文
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
推荐引用方式 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.,...&Zhu, Zuo-Yan.(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.