Please use this identifier to cite or link to this item:
Title: RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing.
Author: Hernández Hernández, Victor
Bueno i Torrens, David, 1965-
Keywords: Genètica animal
Genètica del desenvolupament
Animal genetics
Developmental genetics
Issue Date: 2005
Publisher: Ivyspring International
Abstract: The in vivo 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 in ovo 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 fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. 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 fgf8 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.
Note: Reproducció del document publicat a:
It is part of: International Journal of Biological Sciences, 2005, vol. 1, num. 1, p. 1-12
ISSN: 1449-2288
Appears in Collections:Articles publicats en revistes (Genètica, Microbiologia i Estadística)

Files in This Item:
File Description SizeFormat 
519131.pdf1.04 MBAdobe PDFView/Open

This item is licensed under a Creative Commons License Creative Commons