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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 PublicationDEVELOPMENT GROWTH & DIFFERENTIATION
ISSN0012-1592
Volume47Issue:5Pages:323-331
AbstractDouble-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.
SubtypeArticle
KeywordEsirna No Tail Rnai Sirna Zebrafish
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 AreaCell Biology ; Developmental Biology
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
Indexed BySCI
Language英语
WOS Research AreaCell Biology ; Developmental Biology
WOS SubjectCell Biology ; Developmental Biology
WOS IDWOS:000230301300005
WOS KeywordDOUBLE-STRANDED-RNA ; NO-TAIL ; GENE-EXPRESSION ; MESSENGER-RNA ; MAMMALIAN-CELLS ; FLOOR PLATE ; INJECTION ; DEGRADATION ; KNOCKDOWN ; SPADETAIL
Citation statistics
Cited Times:37[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.ihb.ac.cn/handle/152342/9218
Collection期刊论文
Corresponding AuthorSun, YH, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, 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
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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