Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Journal of Visualized Experiments, 2018 · DOI: doi:10.3791/58171 · Published: September 1, 2018

Regenerative Medicine Neurology Genetics

Simple Explanation

Electroporation is a non-viral method to introduce genetic material into cells. This technique uses electrical pulses to create temporary pores in cell membranes, allowing DNA or RNA to enter. Sensory neurons, which have the ability to regenerate after injury, are used to study axon regeneration. Electroporation is used to manipulate gene expression in these neurons to understand the mechanisms behind axon regeneration. This method allows researchers to study the effects of genes on axon regeneration by either increasing or decreasing their activity. The timing and location of gene manipulation can be controlled, making it a valuable tool for studying axon regeneration.

Study Duration

Not specified

Participants

Six-week-old female CF1 mice weighing 30–35 g

Evidence Level

Not specified

Key Findings

1
The study demonstrates a detailed protocol for in vivo electroporation of adult mouse DRG to manipulate gene expression in sensory neurons.
2
The transfection rate of microRNAs or siRNAs after in vivo electroporation in DRG neurons is close to 90%.
3
The described methodology can also be used as a model to investigate sensory axon regeneration after spinal cord injury.

Research Summary

The study presents a detailed protocol for in vivo electroporation of adult mouse dorsal root ganglion (DRG) to manipulate gene expression in sensory neurons. This approach enables the investigation of mammalian axon regeneration by transfecting plasmids or small RNA oligos into DRGs, allowing for both loss- and gain-of-function experiments. The protocol involves surgical exposure of the DRGs, followed by injection of genetic material and electroporation to facilitate entry into the cells. The method is validated by demonstrating high transfection rates and is used to study axon regeneration after sciatic nerve crush. The in vivo electroporation method offers advantages over viral-based methods, including being less time-consuming and applicable to almost all tissues and cells. However, it has limitations such as a shorter duration of siRNA efficacy and the need for skilled surgical technique.

Practical Implications

Research Tool

Provides a valuable tool for studying the molecular mechanisms regulating mammalian axon regeneration in vivo.

Therapeutic Target Identification

Facilitates the identification of potential therapeutic targets for promoting axon regeneration after nerve injury or spinal cord injury.

Gene Delivery Method

Offers an efficient method for delivering genes or RNA oligos to sensory neurons in vivo, which can be used for gene therapy applications.

Study Limitations

1
The duration of siRNA efficacy to knocking down gene-of-interest is shorter than the virus-based delivery of plasmids.
2
The surgery performed under the microscope requires practice and some manual dexterity.
3
Flattening the sciatic nerve causes overlapping of axons when conducting epifluorescent imaging.

Your Feedback

Was this summary helpful?

Back to Regenerative Medicine