Selective inhibition of gene expression by RNAi in chick embryos in ovo

Selective inhibition of gene expression using double-stranded RNA is a tech nique currently used to study gene function in many biological systems, fro m plants (post-transcriptional gene silencing) to animals. This cellular me chanism, called RNA interference (RNAi), consists of the targeted degradati on 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 dou ble-stranded RNA (dsRNA). It has been postulated that in the case of verteb rates, RNAi mechanisms may not be specific, as the presence of any dsRNA tr iggers a vehement antiviral response mediated by a double-stranded RNA depe ndent protein-kinase, which inhibits transcription and induces apoptosis. S tudies on zebrafish and mouse embryos in which a specific dsRNA was injecte d into the cytoplasm of oocytes, zygotes or blastomers, did not solve the q uestion, 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 d ays later (four days of in ovo development). The whole mount in situ RNA hy bridisation with a fgf8 antisense riboprobe revealed severe inhibition of f gf8 expression and many morphological malformations in organs and structure s where fgf8 is known to be active. We conclude that the RNA interference t echnique could be used in chick embryos in ovoto study the function of gene s involved in development.