Effective gene delivery to adult neurons by a modified form of electroporation

doi:10.1016/j.jneumeth.2004.08.012

Journal of Neuroscience Methods

Volume 142, Issue 1, 15 March 2005, Pages 137-143

https://doi.org/10.1016/j.jneumeth.2004.08.012 Get rights and content

Abstract

Non-viral methods of transfection of cDNAs into adult neurons and other post-mitotic cells are generally very inefficient. However, the recent development of Nucleofector™ technology developed by Amaxa Biosystems allows direct delivery of cDNAs into the nucleus, enabling transfection of non-dividing cells. In this study, we describe a reliable method for culturing large numbers of retinal cells from adult rats and using Nucleofection, we were able to transfect cDNA-encoding GFP (jellyfish green fluorescent protein) into retinal ganglion cells (RGCs) with relatively high efficiency (up to 28%). Neuronal GFP expression was observed within 18 h and continued for up to 14 days. This compares with values up to 60% of RGCs expressing GFP following infection with an HSV-1 vector. Adult rat dorsal root ganglion (DRG) neurons were also successfully transfected. Thus, in summary, Nucleofection provides the possibility for a fast and efficient method for cDNA delivery and study of gene function in adult mammalian neurons.

Introduction

The study of gene function by transfection of foreign cDNAs is widely used in cell biology. However, cDNA transfer into post-mitotic neurons using methods such as calcium phosphate precipitation or cationic lipids is generally inefficient (reviewed by Washbourne and McAllister (2002)). Other methods of gene delivery to neurons using viral vectors or biolistics may be more efficient, but for technical reasons their use has often been limited. For example, biolistic gene delivery requires availability of specialist apparatus, whilst insertion of cDNAs into viral vectors and the subsequent selection and amplification of viral particles is time-consuming and requires appropriate levels of safety precautions.

One technique for gene delivery to neurons which has shown promise is electroporation which utilizes an electric field to create transient pores in cell membranes through which cDNAs may enter from the extracellular medium (reviewed by Inoue and Krumlauf (2001)). The recent development of Nucleofector™ technology allows directed electroporation of cDNAs to the nucleus, thereby enabling transfection of non-dividing cells. This technology has been applied successfully in transfection of several cell types such as dendritic and natural killer cells which are generally difficult to transfect (Lenz et al., 2003, Trompeter et al., 2003). This approach has also been used to transfect embryonic and neonatal chick neurons (B Eickholt, pers. commun.). In the present study, we show that this technology can also be applied to adult neurons, specifically retinal ganglion cells (RGC), with relatively high efficiency.

Section snippets

Plasmids

The plasmid pR19-GFP, containing a cDNA-encoding green fluorescent protein (GFP), was a gift from Dr. Robert Coffin (Biovex and The Windeyer Institute, London).

Preparation of rat retinal ganglion cells

Adult Wistar rats aged 6–8 weeks (purchased from Harlan, UK or bred at King's College London) were killed by inhalation of CO₂, and their retinae dissociated enzymatically using the ‘Papain dissociation system’ (Worthington Biochemical Corporation, New Jersey, USA) with 60 min incubation in papain at 37 °C. Solutions from the dissociation

Rat retinal ganglion cells

Dissociated retinal cells with diameters ≥8 μm (which would include approximately 50% RGC together with other cell types; Guenther et al., 1994) were plated at a density of 1.0 × 10⁵/well. The yield of cells from a single rat was typically sufficient for 15–20 wells. In five experiments, after 3 days in culture, the mean number of attached rounded cells (≥8 μm) was 20,500 ± 2900/well, and of these, 71 ± 5% appeared to be viable on the basis of Calcein^AM labelling, indicating an overall survival

Acknowledgements

This work was supported by the BBSRC and the International Spinal Research Trust (ISRT).

References (13)

L.K. Geiger et al.
Reduced redox state allows prolonged survival of axotomized neonatal retinal ganglion cells
Neuroscience
(2002)
E. Guenther et al.
In vitro identification of retinal ganglion cells in culture without the need of dye labeling
J Neurosci Methods
(1994)
P. Lenz et al.
Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte-derived dendritic cells
FEBS Lett
(2003)
B. Lorber et al.
Effect of lens lesion on neurite outgrowth of retinal ganglion cells in vitro
Mol Cell Neurosci
(2002)
E.C. Ohki et al.
Improving the transfection efficiency of post-mitotic neurons
J Neurosci Methods
(2001)
H.I. Trompeter et al.
Rapid and highly efficient gene transfer into natural killer cells by nucleofection
J Immunol Methods
(2003)

There are more references available in the full text version of this article.

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Effective gene delivery to adult neurons by a modified form of electroporation

Abstract

Introduction

Section snippets

Plasmids

Preparation of rat retinal ganglion cells

Rat retinal ganglion cells

Acknowledgements

Neuroscience

J Neurosci Methods

FEBS Lett

Mol Cell Neurosci

J Neurosci Methods

J Immunol Methods