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Cell Biology JoVE (Open Access) Citable · DOI

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

DOI: 10.3791/1614-v
What you'll learn
  • ✓ Fabricate sharp microinjection pipettes for intranuclear DNA delivery
  • ✓ Perform intranuclear microinjection of cDNA into adult mammalian neurons
  • ✓ Measure heterologous protein function via whole-cell electrophysiology recordings
  • ✓ Achieve high transfection efficiency in post-mitotic cells
Protocol

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Difficulty
advanced
Total time
~2–3 hours per batch of neurons (including cell dissociation, injection setup, and recordings)
Model organism
Adult mammalian neurons (rat SCG neurons)
Biosafety
BSL-1

Steps

1
Fabricate microinjection pipettes from glass

Pull and beveled glass micropipettes to produce sharp, fine-tipped injection needles suitable for penetrating the neuronal nucleus. This requires specialized equipment (pipette puller and microforge) and careful calibration.

▶ 01:39
2
Prepare neurons and set up injection apparatus

Dissociate adult mammalian neurons and plate them on culture dishes. Position the microinjection pipette under a microscope with appropriate micromanipulators and pneumatic pressure control for intranuclear targeting.

▶ 04:54
3
Inject cDNA directly into neuronal nucleus

Penetrate the neuronal nucleus with the loaded micropipette and dispense cDNA construct(s) using controlled pneumatic pressure. Monitor successful injection visually and allow time for protein expression before functional assessment.

▶ 12:00
4
Record whole-cell currents from transfected neurons

Perform patch-clamp whole-cell electrophysiology on injected neurons to measure ion channel function and validate heterologous protein expression in a physiologically relevant context.

▶ 12:00
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