Electroporation-Mediated In Vitro Gene Delivery: An Electric Pulse Technique to Introduce Fluorescent Reporter Protein-Encoding Plasmids Into Cultured Cells
Transkript
For electroporation-based plasmid DNA delivery into primary neuronal cells, begin with a suspension of cells in suitable electroporation media. Add fluorescent protein-encoding plasmid DNA to prepare the electroporation mixture.
Gently transfer the electroporation mixture into an electroporation cuvette, avoiding air bubbles that could negatively affect electroporation efficiency. Transfer the cuvette to the electroporator's cuvette holder; set the electroporation parameters. Initiate electroporation.
The applied high-voltage short electrical pulses across the cell membrane cause differential charge accumulation on either sides of the membrane, increasing the transmembrane potential, the voltage across the cell membrane.
When the induced transmembrane potential exceeds the lipid bilayer breakdown potential, the lipid bilayer destabilizes with the localized configuration change, forming transient hydrophilic pores on the cell membrane. The pores with adequate size provide a pathway for plasmid DNA to enter the cell cytoplasm.
On completion of electroporation, the lipid bilayer reseals, restoring the cell membrane bilayer integrity, thereby entrapping the plasmid DNA. Further, during the phase of cell cycle when the nuclear membrane, the double-layered membrane surrounding the nucleus, disassembles, plasmid DNA translocates to the nucleus.
Plate electroporated cells into media-containing wells of a multi-well plate with protein-coated coverslips. Incubate to allow cells to adhere to the protein-coated coverslip. Successfully transfected cells containing plasmid DNA express the fluorescent proteins.
