A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture
Brudnick Neuropsychiatric Research Institute, Department of Neurobiology; Futai Lab; Graduate School of Biomedical Sciences, Program in Neuroscience; Graduate School of Biomedical Sciences, MD/PhD Program
Investigative Techniques | Laboratory and Basic Science Research | Molecular Biology | Neuroscience and Neurobiology | Research Methods in Life Sciences
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 ( approximately 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. > 10MOmega) and apply high voltage (10V) pulses with high frequency (100Hz) for 1s. 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 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.
Electrophysiology, Gene delivery, Hippocampus, Mouse, Neuron, Organotypic slice culture, Single-cell electroporation
DOI of Published Version
Keener DG, Cheung A, Futai K. A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture. J Neurosci Methods. 2020 May 1;337:108632. doi: 10.1016/j.jneumeth.2020.108632. Epub 2020 Feb 29. PMID: 32126275. Link to article on publisher's site
Journal of neuroscience methods
Keener DG, Cheung A, Futai K. (2020). A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture. Neurobiology Publications. https://doi.org/10.1016/j.jneumeth.2020.108632. Retrieved from https://escholarship.umassmed.edu/neurobiology_pp/257