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.
The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6].
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.
Part 1: Setting up for in utero and ex vivo retinal electroporations
1. For both in utero and ex vivo retinal electroporations
2. For in utero retinal electroporations only
3. For ex vivo retinal electroporations only
Part 2A: In utero retinal electroporation Injecting and electroporating DNA into E14.5 murine retinae in vivo
Part 2B: In utero retinal electroporation Retrieval of electroporated retina at E18.5
Removing retina
Part 3a: Ex vivo retinal electroporation Injecting and electroporating DNA into E14.5 murine retinae in vitro
Part 3b: Ex vivo retinal electroporation Harvesting and plating of GFP+ retinal explants (2 days post-electroporation)
Border Assay
Representative results
In this video, we have demonstrated electroporation techniques, both in utero and ex vivo, for gene delivery into embryonic murine RGCs. In utero retinal electroporation provides a mechanism for manipulating gene expression in RGCs and visualize their axon projections in vivo. Allowing the embryos to survive postnatally permits examination of these GFP+ RGC projections into their target, the lateral geniculate nucleus (LGN). Ex vivo retinal electroporation provides a more controlled ability to target specific retinal regions for use with in vitro assays. Combining in vivo and in vitro analyses derived from these techniques allows thorough characterization of RGC development and axon projections in a subset of RGCs in an otherwise normal background. While our demonstration focuses on RGC axon guidance at the optic chiasm, both in utero and ex vivo retinal electroporations could be beneficial for studying how perturbations in gene expression effect RGC differentiation, dendritic morphology, electrophysiological properties, synapse formation and proper targeting in termination zones such as the LGN and superior colliculus.
We thank Dr. Richard Vallee and Brikha Shrestha for help with the in utero and ex vivo retinal electroporation techniques, respectively, and Dr. T. Sakurai for comments on this protocol. This work was supported by National Institutes of Health Grants F31 NS051008 (T.J.P), the Fondation pour la Recherche Medicale, and the Human Frontier Science Program (A.R.) and R01 EY12736 (C.M.).
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
BTX Electro Square Porator ECM 830 (with foot paddle) | Harvard Apparatus | 450052 | ||
Round tweezer electrodes | Nepa Gene | CUY650-5 or CUY650-7 | ||
Silk Sutures | Henry Schein | 101-2636 | ||
Glass micropipettes with plungers | Drummond | 5-000-1001-X10 | ||
Antibiototic-antimycotic | Invitrogen | 15240096 | ||
DMEM/F12 medium | Gibco | 11330057 | ||
Albumin bovine serum | Sigma | A8806-5G | ||
ITS supplement | Sigma | I-1884 | ||
Penicillin-Streptomycin | Invitrogen | 15140-122 | ||
Methyl cellulose | Sigma | M0512 | ||
Glass Bottom Microwell Dishes | MatTek Corporation | P35G-1.5-14-C | ||
Poly-L-ornithine | Sigma | P4957 | ||
Laminin | Invitrogen | 23017-015 |