Home › Neuroscience › In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors
Neuroscience JoVE (Open Access) Citable · DOI

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

DOI: 10.3791/59465-v
What you'll learn
  • ✓ Produce and purify AAV5 viral vectors for brain injection
  • ✓ Perform intracerebral injection of reprogramming factors into mouse brain
  • ✓ Conduct electrophysiological recordings to assess reprogrammed neuron function
  • ✓ Evaluate maturation and functionality of in vivo reprogrammed interneurons
Protocol

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

Difficulty
advanced
Total time
~4–6 weeks (viral production and purification ~2 weeks; injection and post-operative recovery ~2 weeks; functional analysis ~2 weeks)
Model organism
Mouse (strain not specified, presumed C57BL/6 or equivalent)
Biosafety
BSL-2

Steps

1
Produce AAV5 viral vectors

Generate AAV5 viral vectors carrying reprogramming factors and FLEX-driven GFP reporter using transfection of HEK293 cells or similar production system. Harvest and prepare crude viral lysate for downstream purification.

▶ 01:16
2
Purify AAV5 viral vectors

Concentrate and purify crude AAV5 lysate using column chromatography or iodixanol density gradient centrifugation to obtain high-titer, clean viral stock suitable for intracerebral injection.

▶ 03:41
3
Inject reprogramming factors into brain

Perform stereotactic intracerebral injection of purified AAV5 vectors into target brain region of anesthetized mice. Allow post-operative recovery and transgene expression over 2–3 weeks.

▶ 06:01
4
Conduct electrophysiological recordings

Perform whole-cell or cell-attached patch-clamp electrophysiology on GFP+ reprogrammed cells in acute brain slices to characterize membrane properties, firing patterns, and synaptic integration.

▶ 08:34
5
Assess maturation and functionality of reprogrammed neurons

Analyze electrophysiological data and morphological features to confirm that reprogrammed cells exhibit functional neuron properties and progressive maturation over time post-injection.

▶ 10:33
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