A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture.
Electrophysiology
Gene delivery
Hippocampus
Mouse
Neuron
Organotypic slice culture
Single-cell electroporation
Journal
Journal of neuroscience methods
ISSN: 1872-678X
Titre abrégé: J Neurosci Methods
Pays: Netherlands
ID NLM: 7905558
Informations de publication
Date de publication:
01 05 2020
01 05 2020
Historique:
received:
13
11
2019
revised:
19
01
2020
accepted:
10
02
2020
pubmed:
4
3
2020
medline:
22
6
2021
entrez:
4
3
2020
Statut:
ppublish
Résumé
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. 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. Our method achieves a high transfection efficiency (∼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. The majority of existing methods use fine-tipped glass electrodes (i.e. > 10 MΩ) 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. 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.
Sections du résumé
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 (∼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 MΩ) 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 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.
Identifiants
pubmed: 32126275
pii: S0165-0270(20)30054-6
doi: 10.1016/j.jneumeth.2020.108632
pmc: PMC7521366
mid: NIHMS1575981
pii:
doi:
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
108632Subventions
Organisme : NINDS NIH HHS
ID : R01 NS085215
Pays : United States
Organisme : NIGMS NIH HHS
ID : T32 GM107000
Pays : United States
Informations de copyright
Copyright © 2020 Elsevier B.V. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Competing Interest The authors declare that they have no conflicts of interest.
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