A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture
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Student Authors
Amy CheungDavid G. Keener
Academic Program
MD/PhDUMass Chan Affiliations
Graduate School of Biomedical Sciences, MD/PhD ProgramGraduate School of Biomedical Sciences, Neuroscience Program
Futai Lab
Brudnick Neuropsychiatric Research Institute
Neurobiology
Document Type
Journal ArticlePublication Date
2020-05-01Keywords
ElectrophysiologyGene delivery
Hippocampus
Mouse
Neuron
Organotypic slice culture
Single-cell electroporation
Investigative Techniques
Laboratory and Basic Science Research
Molecular Biology
Neuroscience and Neurobiology
Research Methods in Life Sciences
Metadata
Show full item recordAbstract
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.Source
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
DOI
10.1016/j.jneumeth.2020.108632Permanent Link to this Item
http://hdl.handle.net/20.500.14038/37985PubMed ID
32126275Related Resources
ae974a485f413a2113503eed53cd6c53
10.1016/j.jneumeth.2020.108632