Selective inhibition of gene expression by RNAi in chick embryos in ovo
Published: 1 June 2001
V Hernandez-Hernandez, J Fernandez, A Cardona, R Romero, D Bueno
Univ Barcelona, Fac Biol, Dept Genet, E-08028 Barcelona, Catalonia, Spain
Selective inhibition of gene expression using double-stranded RNA is a technique currently used to study gene function in many biological systems, from plants (post-transcriptional gene silencing) to animals. This cellular mechanism, called RNA interference (RNAi), consists of the targeted degradation of the mRNA into 21-23 nucleotide fragments by the action of a specific endonuclease complex, and is directed by the presence of the homologous double-stranded RNA (dsRNA). It has been postulated that in the case of vertebrates, RNAi mechanisms may not be specific, as the presence of any dsRNA triggers a vehement antiviral response mediated by a double-stranded RNA dependent protein-kinase, which inhibits transcription and induces apoptosis. Studies on zebrafish and mouse embryos in which a specific dsRNA was injected into the cytoplasm of oocytes, zygotes or blastomers, did not solve the question, as the information about mutant phenotypes obtained was difficult to understand, causing some contradictory reports. To clarify this question we microinjected chicken fibroblast growth factor-8 (fgf8) dsRNA into the neural tube of 2-day-old chick embryos which were killed and analysed two days later (four days of in ovo development). The whole mount in situ RNA hybridisation with a fgf8 antisense riboprobe revealed severe inhibition of fgf8 expression and many morphological malformations in organs and structures where fgf8 is known to be active. We conclude that the RNA interference technique could be used in chick embryos in ovoto study the function of genes involved in development.