Double-stranded RNA injection produces nonspecific defects in zebrafish.
We have investigated the ability of dsRNA to inhibit gene functions in zebrafish using sequences targeted to the maternal gene pouII-1, the transgene GFP, and an intron of the zebrafish gene terra. We found that embryos injected with all of these dsRNAs at approximately 7.5 pg/embryo or higher had general growth arrest during gastrulation and displayed various nonspecific defects at 24 h postfertilization, although embryonic development was unaffected before the midblastula stage. Reducing dsRNA concentration could alleviate the global defects. Injection of GFP dsRNA (7.5-30 pg/embryo) did not inhibit GFP expression in transgenic fish, although abnormal embryos were induced. Co-injection of GFP mRNA with either GFP or non-GFP dsRNA caused reduction of GFP expression. Whole-mount in situ hybridization clearly showed that embryos injected with dsRNA degraded co-injected and endogenous mRNA without sequence specificity, indicating that dsRNA has a nonspecific effect at the posttranscriptional level. It appears that RNAi is not a viable technique for studying gene function in zebrafish embryos.
Zhao
Z
Z
Institute of Cellular and Developmental Biology, Tsinghua University, Beijing, 100084, China.
Cao
Y
Y
Li
M
M
Meng
A
A
D000015:Abnormalities, Multiple / Q000139:chemically induced*; D000818:Animals; D030801:Animals, Genetically Modified; D004268:DNA-Binding Proteins / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins; D017403:In Situ Hybridization; D008164:Luminescent Proteins / Q000235:genetics; D008845:Microinjections; D050814:Octamer Transcription Factor-3; D020871:RNA Stability; D016372:RNA, Antisense / Q000633:toxicity; D012330:RNA, Double-Stranded / Q000633:toxicity*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D014157:Transcription Factors / Q000235:genetics; D019076:Transgenes; D015027:Zebrafish; D029961:Zebrafish Proteins
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D050814:Octamer Transcription Factor-3; C085437:Pou5f3 protein, zebrafish; D016372:RNA, Antisense; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D029961:Zebrafish Proteins; C486539:dmrt2a protein, zebrafish; D049452:Green Fluorescent Proteins