PUBLICATION
Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo
- Authors
- Oates, A.C., Bruce, A.E., and Ho, R.K.
- ID
- ZDB-PUB-000824-12
- Date
- 2000
- Source
- Developmental Biology 224(1): 20-28 (Journal)
- Registered Authors
- Bruce, Ashley, Ho, Robert K., Oates, Andrew
- Keywords
- spadetail; tbx16; no tail; nieuwkoid; dsRNA; RNAi; zebrafish; embryogenesis
- MeSH Terms
-
- Animals
- Embryo, Nonmammalian
- Gene Silencing
- In Situ Hybridization
- Microinjections
- Phenotype
- RNA, Double-Stranded/genetics*
- RNA, Messenger/analysis
- T-Box Domain Proteins/genetics
- T-Box Domain Proteins/metabolism*
- Zebrafish/embryology
- Zebrafish/genetics*
- Zebrafish Proteins*
- PubMed
- 10898958 Full text @ Dev. Biol.
Citation
Oates, A.C., Bruce, A.E., and Ho, R.K. (2000) Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo. Developmental Biology. 224(1):20-28.
Abstract
We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of dsRNA corresponding to the T-box gene tbx16/spadetail (spt) into early wild-type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the blastula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/spt function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffected by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of injecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Brachyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h postfertilization. In blind studies, we could not distinguish between the effects of the various dsRNAs. Consistent with a common effect of dsRNA, regardless of sequence, injection of dsRNA from the lacZ gene was likewise effective in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current methodology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping