Single-Cell Electroporation of Neurons.
Single-cell electroporation allows the transfection of a small number of neurons in an organotypic culture with a single plasmid or a defined mixture of plasmids. Desired protein expression levels can vary depending on the experimental goals (e.g., high expression levels are needed for optogenetic experiments); however, when too much protein is expressed, cellular toxicity and cell death may arise. To a large degree, protein expression can be controlled by adjusting the concentration of plasmid DNA in the electroporation pipette. Here, we present a protocol for transfecting individual neurons in hippocampal slice cultures by electroporation. Essentially, a patch-clamp setup is required that includes an upright microscope with infrared differential interference contrast or Dodt contrast with a camera and a specialized amplifier that is able to deliver large-voltage pulses to the electroporation pipette.
Institute for Synaptic Physiology, Center for Molecular Neurobiology (ZMNH), 20251 Hamburg, Germany.
J Simon
JS
Wiegert
Institute for Synaptic Physiology, Center for Molecular Neurobiology (ZMNH), 20251 Hamburg, Germany.
Christine E
0000-0003-0345-3665
CE
Gee
Institute for Synaptic Physiology, Center for Molecular Neurobiology (ZMNH), 20251 Hamburg, Germany thomas.oertner@zmnh.uni-hamburg.de.
Thomas G
0000-0002-2312-7528
TG
Oertner
D000818:Animals; D018274:Electroporation / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors; D006624:Hippocampus / Q000502:physiology; D051379:Mice; D009474:Neurons / Q000502:physiology*; D010957:Plasmids; D051381:Rats; D059010:Single-Cell Analysis; D014162:Transfection / Q000379:methods*; D019076:Transgenes