In ovo electroporation of miRNA plasmids to silence genes in a temporally and spatially controlled manner

The ability to spatially and temporally control gene expression during development is crucial for the elucidation of gene function in vivo. The use of RNA interference (RNAi)-based technologies in combination with oviparous animal models allows for efficient, precise gene silencing. We have developed approaches using RNAi in the chicken embryo to analyze gene function during neural tube development. Here we describe the construction of plasmids that direct the expression of one or two artificial microRNAs (miRNAs) to knock down endogenous target protein/s upon electroporation into the spinal cord. The miRNA cassette is directly linked to a fluorescent protein reporter, which allows the faithful visualization of transfected cells. Different promoters/enhancers drive transcript expression in genetically defined cell subpopulations in the neural tube. Mixing multiple RNAi vectors allows combinatorial knockdowns of two or more genes in different cell types, thus permitting the rapid analysis of complex cellular and molecular interactions.