Gene Expression via Single-Cell Electroporation in Mouse Hippocampal Slice Cultures

Begin with a hippocampal slice from a mouse brain cultured on a culture insert in a dish.

Cut the insert containing the slice and secure it in an electroporation chamber under a microscope.

Perfuse the chamber with a buffer containing a voltage-gated sodium channel blocker to inhibit overexcitation and prevent cellular toxicity.

Position a pipette containing plasmids with the gene of interest near the slice.

Maintain positive pressure to prevent pipette clogging while approaching a target hippocampal neuron until a membrane dimple forms.

Switch to negative pressure to create a seal with the membrane.

Alternate between positive and negative pressure to minimize cellular damage.

Apply an electroporation pulse to transiently permeabilize the membrane, facilitating plasmid entry.

Withdraw the pipette while maintaining positive pressure, and repeat electroporation for neighboring target neurons.

Transfer the electroporated slice to a fresh insert and incubate it to allow expression of the gene of interest.

To prepare slice cultures for electroporation, transfer the culture inserts of interest into individual three centimeter petri dishes loaded with 900 microliters of culture medium, and place th