Ex Vivo Electroporation of Chick Embryo Cerebellar Slices: A Method to Introduce Plasmid DNA Encoding Green Fluorescent Protein to Visualize Granular Cell Development
Transcrição
Cerebellar granule cells are the brain's most abundant type of neurons. The external granular layer, EGL, is composed solely of granular cell precursors, GCPs, that differentiate and migrate inwardly to form the inner granular layer, IGL.
To visualize granular cell development, prep an electroporation chamber by attaching an anode to the base of a Petri dish. Add culture medium to cover the electrode. Now, place a culture insert on the medium-covered electrode. Transfer the chick embryo cerebellum slices onto the culture insert in a sagittal orientation.
The sagittal plane allows the target layer-EGL to be identified against different cerebellum layers. Add a viscous dye solution containing a GFP plasmid DNA over EGL. The GFP plasmid DNA encodes for a green fluorescent protein that distributes homogeneously within the cell cytoplasm and enables live-cell imaging.
Next, place a cathode in the medium near EGL but not directly on the tissue. Then, apply multiple electric pulses to different EGL regions. The electric pulses induce temporary pores in the cell membrane, allowing cells to take up the plasmid DNA. Subsequently, plasmid DNA enables GFP expression in granular cells.
Image the cerebellar slices under a fluorescence microscope to observe granular cell differentiation and migration pattern across different stages of cell development.
