In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

The aim of this procedure is to assess axon guidance defects in the chicken neural tube after downregulating gene expression in a cell type specific traceable manner. This is accomplished by preparing fertilized chicken eggs for innovo electroporation by cutting a window in the eggshell. The second step is to inject and electro operate DNA constructs encoding microRNAs into the neural tube.

Next, the electro rated spinal cords are dissected and fixed in flat mount open book preparations. The final step is to inject a fluorescent dye to visualize the axonal projections. Ultimately, fluorescent microscopy is used to show both the cells experiencing gene silencing and to assess the subsequent effects on commiss axon guidance.