| Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase | |
| Liu, WY; Wang, Y; Sun, YH; Wang, Y; Wang, YP; Chen, SP; Zhu, ZY; Sun, YH, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China | |
| 2005-06-01 | |
| Source Publication | DEVELOPMENT GROWTH & DIFFERENTIATION
 |
| ISSN | 0012-1592 |
| Volume | 47Issue:5Pages:323-331 |
| Abstract | 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.; 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. |
| Subtype | Article |
| Keyword | Esirna No Tail Rnai Sirna Zebrafish |
| Department | 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 |
| Subject Area | Cell Biology ; Developmental Biology |
| WOS Headings | Science & Technology ; Life Sciences & Biomedicine |
| Indexed By | SCI |
| Language | 英语 |
| WOS Research Area | Cell Biology ; Developmental Biology |
| WOS Subject | Cell Biology ; Developmental Biology |
| WOS ID | WOS:000230301300005 |
| WOS Keyword | DOUBLE-STRANDED-RNA ; NO-TAIL ; GENE-EXPRESSION ; MESSENGER-RNA ; MAMMALIAN-CELLS ; FLOOR PLATE ; INJECTION ; DEGRADATION ; KNOCKDOWN ; SPADETAIL |
| Citation statistics | |
| Document Type | 期刊论文 |
| Identifier | http://ir.ihb.ac.cn/handle/152342/9218 |
| Collection | 期刊论文 |
| Corresponding Author | Sun, YH, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China |
| Affiliation | 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 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.,...&Sun, YH, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China.(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. |
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