Double–Stranded RNA Induces Specific Developmental Defects in Zebrafish Embryos

Wargelius, Anna; Ellingsen, Ståle; Fjose, Anders

doi:10.1006/bbrc.1999.1343

Cited by 173 publications

(109 citation statements)

References 17 publications

Supporting

Mentioning

105

Contrasting

Order By: Relevance

“…To interfere with Vasa protein levels in the early zebrafish embryo, we applied RNAi (Wargelius et al, 1999), which has been successfully applied in different organisms (Fraser et al, 2000;Misquitta and Paterson, 1999). We injected vasa dsRNA into one-or two-cellstage embryos that were raised to 24 hpf (hours postfertilization) and then were evaluated for the presence of vasa mRNA or Vasa protein by whole-mount in situ hybridization and whole-mount immunostaining, respectively.…”

Section: Resultsmentioning

confidence: 99%

A zebrafish vasa morphant abolishes Vasa protein but does not affect the establishment of the germline

Braat

Water

Korving

et al. 2001

Genesis

View full text Add to dashboard Cite

Section: Resultsmentioning

confidence: 99%

A zebrafish vasa morphant abolishes Vasa protein but does not affect the establishment of the germline

Braat

Water

Korving

et al. 2001

Genesis

View full text Add to dashboard Cite

“…A few years ago, RNA interference seemed to be a promising new approach for specifically inactivating genes in zebrafish (Wargelius et al 1999;Li et al 2000), but success rates in zebrafish were low and varying, with reports of major nonspecific effects on embryonic development reported as well (Oates et al 2000;Zhao et al 2001). The most widely used reverse genetics method in zebrafish is undoubtedly the use of morpholinos that are targeted to the translation initiation site of a specific transcript, thereby inhibiting its translation (Nasevicius and Ekker 2000).…”

mentioning

confidence: 99%

Efficient Target-Selected Mutagenesis in Zebrafish

Wienholds

Eeden²,

Kosters³

et al. 2003

Genome Research

412

296

View full text Add to dashboard Cite

One of the most powerful methods available to assign function to a gene is to inactivate or knockout the gene. Recently,we described the first target-selected knockout in zebrafish. Here,we report on the further improvements of this procedure,resulting in a highly efficient and easy method to do target-selected mutagenesis in zebrafish. A library of 4608 ENU-mutagenized F1 animals was generated and kept as a living stock. The DNA of these animals was screened for mutations in 16 genes by use of CEL-I-mediated heteroduplex cleavage (TILLING) and subsequent resequencing. In total,255 mutations were identified,of which 14 resulted in a premature stop codon,7 in a splice donor/acceptor site mutation,and 119 in an amino acid change. By this method,we potentially knocked out 13 different genes in a few months time. Furthermore,we show that TILLING can be used to detect the full spectrum of ENU-induced mutations in a vertebrate genome with the presence of many naturally occurring polymorphisms

show abstract

“…Some findings suggested that dsRNA-mediated interference could be used to analyze the functional roles of genes in zebrafish, and the interference of gene function showed a strong dependence on the amount of dsRNA (Wargelius et al 1999;Acosta et al 2005). Other reports indicated that dsRNA had a nonspecific effect at the posttranscriptional level, and it seemed that RNAi was not a viable technique for studying gene function in zebrafish embryos (Oates et al 2000;Li et al 2000;Zhao et al 2001;Mangos et al 2001).…”

Section: Discussionmentioning

confidence: 99%

The Cytomegalovirus Promoter-Driven Short Hairpin RNA Constructs Mediate Effective RNA Interference in Zebrafish In Vivo

Zhang

Wang

et al. 2008

Mar Biotechnol

View full text Add to dashboard Cite

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNAgenerating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05±1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30±4% and decreased the level of their corresponding mRNAs up to 54.52 ± 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

show abstract

Double–Stranded RNA Induces Specific Developmental Defects in Zebrafish Embryos

Cited by 173 publications

References 17 publications

A zebrafish vasa morphant abolishes Vasa protein but does not affect the establishment of the germline

A zebrafish vasa morphant abolishes Vasa protein but does not affect the establishment of the germline

Efficient Target-Selected Mutagenesis in Zebrafish

The Cytomegalovirus Promoter-Driven Short Hairpin RNA Constructs Mediate Effective RNA Interference in Zebrafish In Vivo

Contact Info

Product

Resources

About