Journal
JOURNAL OF NEUROSCIENCE METHODS
Volume 337, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.jneumeth.2020.108632
Keywords
Single-cell electroporation; Neuron; Gene delivery; Electrophysiology; Organotypic slice culture; Mouse; Hippocampus
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Funding
- National Institutes of Health [R01NS085215, T32 GM107000]
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Background: 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. New method: We have developed an electroporation technique that combines the use of large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses. Here, we describe the technique and compare it with existing methods. Results: Our method achieves a high transfection efficiency (similar to 80%) in both excitatory and 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. In addition, we demonstrate the ability to transfect different genes into neighboring cells. Comparison with existing methods: The majority of existing methods use fine-tipped glass electrodes (i.e. > 10 MO) and apply high voltage (10 V) pulses with high frequency (100 Hz) for 1 s. These parameters contribute to practical difficulties thus lowering the transfection efficiency. Our unique method minimizes electrode clogging and therefore procedure duration, increasing transfection efficiency and cellular viability. Conclusions: Our modifications, relative to current methods, optimize electroporation efficiency and cell sur-vival. 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.
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