RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by <italic>fgf8</italic> silencing

• Format: Article
• Language: English
• Published: Ivyspring International Publisher 2005-01-01
• Series: International Journal of Biological Sciences
• Subjects: RNA interference (RNAi); small interfering RNA (siRNA); short hairpin RNA (shRNA); chick embryo; isthmus; <italic>fgf8</italic>
• Online Access: http://www.biolsci.org/v01p0001.htm

<p>The <italic>in vivo</italic> accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through <italic>in ovo</italic> electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on <italic>fgf8</italic> expression in the developing isthmus, we show that no morphological defects are observed, and that <italic>fgf8</italic> expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, <italic>fgf8</italic> expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to <italic>fgf8</italic>. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which <italic>fgf8</italic> gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.</p>