Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase
文献类型：期刊论文
| 作者 | Liu, WY; Wang, Y; Sun, YH; Wang, Y; Wang, YP; Chen, SP; Zhu, ZY |
| 刊名 | DEVELOPMENT GROWTH & DIFFERENTIATION
 |
| 出版日期 | 2005-06-01 |
| 卷号 | 47期号:5页码:323-331 |
| ISSN号 | 0012-1592 |
| 关键词 | esiRNA no tail RNAi siRNA zebrafish |
| 通讯作者 | Sun, YH, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China |
| 中文摘要 | Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 mu g/mu L. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics. |
| 英文摘要 | Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 mu g/mu L. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics. |
| 学科主题 | Cell Biology; Developmental Biology |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 类目[WOS] | Cell Biology ; Developmental Biology |
| 研究领域[WOS] | Cell Biology ; Developmental Biology |
| 关键词[WOS] | DOUBLE-STRANDED-RNA ; NO-TAIL ; GENE-EXPRESSION ; MESSENGER-RNA ; MAMMALIAN-CELLS ; FLOOR PLATE ; INJECTION ; DEGRADATION ; KNOCKDOWN ; SPADETAIL |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000230301300005 |
| 公开日期 | 2010-10-13 |
| 源URL | [http://ir.ihb.ac.cn/handle/152342/9218]  |
| 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 | Liu, WY,Wang, Y,Sun, YH,et al. Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase[J]. DEVELOPMENT GROWTH & DIFFERENTIATION,2005,47(5):323-331. |
| APA | Liu, WY.,Wang, Y.,Sun, YH.,Wang, Y.,Wang, YP.,...&Zhu, ZY.(2005).Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase.DEVELOPMENT GROWTH & DIFFERENTIATION,47(5),323-331. |
| MLA | Liu, WY,et al."Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase".DEVELOPMENT GROWTH & DIFFERENTIATION 47.5(2005):323-331. |
入库方式： OAI收割
来源：水生生物研究所
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