In utero electroporation (IUE) of plasmid DNA into specific brain regions such as hippocampus and cortex has made it possible to reduce protein expression levels or even replace the endogenous protein with site-directed mutant proteins to reveal important physiological consequences. For example, shRNA can mediate targeted knockdown, and can be complemented by simultaneously expressing the shRNA immune wild-type protein to validate on-target effects, or by expression of an shRNA-immune protein harboring site-specific mutations. More recently, IUE has been adapted to express CRISPR/Cas9 components for targeted gene editing that abolishes protein expression. Utilizing these approaches via IUE results in transfected neurons that are interspersed with non-transfected control neurons in the same brain slices, allowing for direct comparisons. Because IUE is performed late in embryonic development and is confined to a relatively small percentage of neurons, developmental compensatory mechanisms that may compromise gene targeting via germ line transmission are minimized. Thus, IUE presents a powerful opportunity to rapidly and cost-effectively dissect molecular and cellular physiology in the context of the intact brain.
- Potassium channel
- In utero electroporation