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Neuroscience JoVE (Open Access) Citable · DOI

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

DOI: 10.3791/1333-v
What you'll learn
  • ✓ Perform in utero retinal electroporation to manipulate RGC gene expression in developing mouse embryos
  • ✓ Execute ex vivo retinal electroporation on isolated E14.5 retinae using DNA injection and electric pulses
  • ✓ Retrieve and culture electroporated retinal tissue to assess gene expression and RGC properties
Protocol

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

Difficulty
advanced
Total time
~4-5 days (embryonic manipulation at E14.5, retrieval at E18.5, ex vivo culture 2 days post-electroporation)
Model organism
Mouse (embryonic, E14.5-E18.5)
Biosafety
BSL-1

Steps

1
Introduction to retinal electroporation techniques

Overview of in utero and ex vivo electroporation methods for manipulating RGC gene expression and examining developmental effects on axon guidance and functional properties.

▶ 01:07
2
Inject and electroporate DNA into E14.5 retinae in vivo

Perform surgical exposure of murine embryonic retinae at E14.5 gestation, microinject plasmid DNA, and apply electrical pulses to achieve transfection of retinal ganglion cells.

▶ 01:46
3
Retrieve electroporated retina at E18.5 embryonic stage

Dissect and isolate retinal tissue from electroporated embryos at E18.5, 4 days post-electroporation, for downstream analysis of gene expression.

▶ 08:40
4
Inject and electroporate DNA into isolated E14.5 retinae

Extract E14.5 retinae in vitro, inject plasmid DNA via micropipette, and apply electric pulses to transfect retinal cells under controlled ex vivo conditions.

▶ 12:34
5
Harvest and culture GFP+ retinal explants post-electroporation

Culture electroporated retinal tissue for 2 days, identify GFP-positive cells, and plate explants for further analysis of RGC development and properties.

▶ 18:49
6
Analyze representative results from both approaches

Review fluorescent imaging and functional outcomes from in utero and ex vivo electroporation to demonstrate successful gene expression manipulation in RGCs.

▶ 23:33
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