Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase.
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 microg/microL. 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.
Liu
Wei-Yi
WY
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Wang
Yan
Y
Sun
Yong-Hua
YH
Wang
Yun
Y
Wang
Ya-Ping
YP
Chen
Shang-Ping
SP
Zhu
Zuo-Yan
ZY
D000818:Animals; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005326:Fetal Proteins; D019204:GTP-Binding Proteins / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D008845:Microinjections; D008969:Molecular Sequence Data; D010641:Phenotype; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics*; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism; D000098908:Brachyury Protein
D012321:DNA-Directed RNA Polymerases; D005326:Fetal Proteins; D019204:GTP-Binding Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D020825:T-Box Domain Proteins; D029961:Zebrafish Proteins; D000098908:Brachyury Protein; C070852:RNA polymerase SP6