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    A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture

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    Authors
    Keener, David G.
    Cheung, Amy
    Futai, Kensuke
    Student Authors
    Amy Cheung
    David G. Keener
    Academic Program
    MD/PhD
    UMass Chan Affiliations
    Graduate School of Biomedical Sciences, MD/PhD Program
    Graduate School of Biomedical Sciences, Neuroscience Program
    Futai Lab
    Brudnick Neuropsychiatric Research Institute
    Neurobiology
    Document Type
    Journal Article
    Publication Date
    2020-05-01
    Keywords
    Electrophysiology
    Gene 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
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    Link to Full Text
    https://doi.org/10.1016/j.jneumeth.2020.108632
    Abstract
    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.2020.108632
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/37985
    PubMed ID
    32126275
    Related Resources

    Link to Article in PubMed

    ae974a485f413a2113503eed53cd6c53
    10.1016/j.jneumeth.2020.108632
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    Morningside GSBS Scholarly Publications
    Neurobiology Student Publications
    Neurobiology Faculty Publications

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