Home › Neuroscience › Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening
Neuroscience JoVE (Open Access) Citable · DOI

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

DOI: 10.3791/50146-v
What you'll learn
  • ✓ Perform in utero electroporation to introduce genes into rat cortex/hippocampus at E16
  • ✓ Use bioluminescence imaging with luciferase to verify transfection success postnatally
  • ✓ Analyze and quantify luminescence signals from electroporated brain regions
  • ✓ Design targeted neuronal modifications for behavioral and neuropathology studies
Protocol

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

Difficulty
advanced
Total time
~2–3 weeks (IUE procedure ~30 min per animal; imaging begins postnatal day 3–5; analysis ongoing)
Model organism
Rat (Sprague Dawley or equivalent)
Biosafety
BSL-1

Steps

1
Perform in utero electroporation at E16

Surgically access pregnant rat uterus and deliver plasmid DNA into lateral ventricles of embryonic cortex/hippocampus. Apply electrical pulses to transfect target neurons with transgene and luciferase reporter.

▶ 02:01
2
Conduct postnatal bioluminescence live imaging

After birth, administer luciferin substrate and use sensitive camera/photomultiplier to detect bioluminescence emission from luciferase-expressing neurons in anesthetized pups. Image at postnatal days 3–5 or later.

▶ 03:10
3
Analyze bioluminescence images quantitatively

Define regions of interest over electroporated brain areas and measure photon counts, signal-to-background ratio, and spatial distribution. Confirm successful transfection by comparing signal intensity to non-electroporated controls.

▶ 05:47
4
Validate results and proceed to behavior studies

Confirm robust luminescence detection in target regions indicates reliable gene expression. Use successfully transfected animals for downstream behavioral testing or neuropathological analysis.

▶ 06:41
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