Abstract Details

Exogenous gene introduction by transfection is one of the most important approaches for understanding the function of specific genes at the cellular level. Electroporation has a long-standing history as a versatile gene delivery technique in vitro and in vivo. However, it has been underutilized in vitro because of technical difficulty and insufficient transfection efficiency.

The aim of the present study was to optimize a single-cell electroporation method for in vitro gene transfer across different cell types that can be performed with existing electrophysiology equipment and techniques.

We have developed an electroporation technique that combines the use of large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses to deliver genes of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons.

Our method achieves a high transfection efficiency (∼80%) in both excitatory and different classes of inhibitory neurons with no detectable side effects on their function. We demonstrate this method is capable of transferring at least three different genes into a single neuron and transfecting different genes into neighboring cells. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages.

Our modifications, relative to current methods, optimize electroporation efficiency and cell survival. Our approach offers distinct research strategies not only in elucidating cell-autonomous functions of genes but also for assessing genes contributing to intercellular functions, such as trans-synaptic interactions.