citations

                    Optimization of adult sensory neuron electroporation to study mechanisms of neurite growth
                

Authors:

McCall J, Nicholson L, Weidner N, Blesch A.

In:

Source: Frontiers in Molecular Neuroscience
Publication Date: (2012)
Issue: 5: (8)

Research Area:

Neurobiology

Cells used in publication:

Dorsal root gang. (DRG), rat: Species: rat; Tissue Origin: brain

Platform:

4D-Nucleofector® X-Unit

Experiment

Summary (by Lonza): Primary adult dorsal root ganglia neurons (DRGs) are an ideal model for studying many neuronal functions because they survive in culture in an adult stage while most other mammalian neuron types that can be kept in culture for longer time are isolated from embryonic or early postnatal brain. Aim of this study was to find an electroporation protocol for adult DRGs that provides efficient transfection of larger plasmids to study neurite outgrowth, limits the number of required cells and minimizes the use of serum. For that purpose the authors first compared Invitrogen’s Neon Transfection System with Lonza’s 4D-Nucleofector™ System for transfection of a large plasmid (approx.. 10 kb) into primary rat DRGs. After testing several transfection parameters on each device, the authors found that the 4D-Nucleofector™ X Unit was at least 3 times better than the Neon in terms of transfection efficiency (39-42%) and gave slightly better cell viability. Subsequent experiments with 4D-Nucleofector™ X Unit also showed that cell numbers can be reduced to 3x105 cells (in 20 µl) with 2 µg DNA and cells can be kept serum-free post transfection.

Abstract

The development of eukaryotic transfection technologies has been rapid in recent years, providing the opportunity to better analyze cell-autonomous mechanisms influencing various cellular processes, including cell-intrinsic regulators of regenerative neurite growth and survival. Electroporation is one of the more effective methodologies for transfection of post-mitotic neurons demonstrating sufficient neuronal survival and transfection efficiency. To further maximize the number of transfected neurons especially with large plasmids, to limit the cellular exposure to serum, and to minimize the number of animals required for cell isolation per experiment, we compared two state-of-the-art electroporation devices for in vitro transfection of adult rat dorsal root ganglion (DRG) neuron cultures. By refining different parameters, transfection efficiencies of 39-42% could be achieved using the Lonza 4D-Nucleofector X-unit system, 1.5-2-fold higher rates than those that have been previously published for adult DRG neurons using smaller plasmid sizes. Our protocol further limits the number of cells required to 3 × 10(5) cells per 20 ?l reaction using only 2 ?g DNA/reaction and allows for the complete omission of serum post-transfection. Application of this optimized protocol will contribute to furthering the study of neuron-intrinsic mechanisms responsible for growth and survival under physiological and pathophysiological conditions.