Double-stranded RNA injection produces null phenotypes in zebrafish.
Zebrafish is a simple vertebrate that has many attributes that make it ideal for the study of developmental genetics. One feature that has been lacking in this model system is the ability to disable specifically targeted genes. Recently, double-stranded RNA has been used to silence gene expression in the nematode Caenorhabditis elegans. We have found that expression of the green fluorescent protein (GFP) from a microinjected plasmid vector can be suppressed in zebrafish embryos by the coinjection of a double-stranded RNA that is specifically targeted to GFP. To determine that double-stranded RNA can attenuate endogenous gene expression, single-cell zebrafish embryos were injected with double-stranded RNA specifically targeted to Zf-T and Pax6.1. We found that microinjection of double-stranded Zf-T RNA resulted in a high incidence of a phenotype similar to that of ntl. Furthermore, Zf-T gene expression could not be detected by in situ hybridization and the message was decreased by 75% by semiquantitative RT-PCR in 12-h embryos that had been injected with the double-stranded RNA. Expression of the zebrafish genes sonic hedgehog and floating head was altered in the embryos microinjected with the Zf-T double-stranded RNA in a manner that is remarkably similar to the zebrafish no-tail mutant. Microinjection of double-stranded RNA targeted to Pax6.1 was associated with depressed expression of Pax6. 1 and resulted in absent or greatly reduced eye and forebrain development, similar to the phenotype seen in mouse mutants. Simultaneous injection of Pax6.1 and Zf-T resulted in embryos lacking notochords, eyes, and brain structures.
Li
Y X
YX
Developmental Biology Program, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, 30912-2640, USA.
Farrell
M J
MJ
Liu
R
R
Mohanty
N
N
Kirby
M L
ML
D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004626:Embryology / Q000379:methods; D005136:Eye Proteins; D005326:Fetal Proteins; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D020868:Gene Silencing; D017930:Genes, Reporter; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins; D053823:Hedgehog Proteins; D018398:Homeodomain Proteins / Q000096:biosynthesis; D017403:In Situ Hybridization; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008845:Microinjections; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D010641:Phenotype; D014176:Protein Biosynthesis; D012330:RNA, Double-Stranded / Q000494:pharmacology*; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020825:T-Box Domain Proteins / Q000235:genetics*; D013623:Tail / Q000002:abnormalities; D015534:Trans-Activators; D014157:Transcription Factors / Q000096:biosynthesis; D015027:Zebrafish / Q000196:embryology*; D029961:Zebrafish Proteins; D000098908:Brachyury Protein
D004268:DNA-Binding Proteins; D005136:Eye Proteins; D005326:Fetal Proteins; D049452:Green Fluorescent Proteins; D053823:Hedgehog Proteins; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012330:RNA, Double-Stranded; D012097:Repressor Proteins; D020825:T-Box Domain Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D029961:Zebrafish Proteins; D000098908:Brachyury Protein; C000605732:Pax6 protein, mouse; C096660:noto protein, zebrafish