Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Summary

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Abstract

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase^1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.

Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina^5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation⁶.

In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Protocol

1. Equipment Set-up for Electroporation

1. Electrodes: We modified Dumont #5 forceps to use as electrodes.
   1. Separate and break apart the forceps.
   2. Solder a wire at the wider end of each prong. Wrap the wire attached and prongs with insulation tape leaving approximately 25-30 mm of the tip of the prongs exposed.
   3. Put the modified forceps back together with any suitable plastic spacer (e.g a button) between the two prongs to provide spring action.
2. Electrical Equipment: We use an electrical stimulator to deliver current pulses for electroporation and an oscilloscope and....

Discussion

In this video we demonstrate an in vivo electroporation protocol that results in labeling of single or small clusters of retinal neurons in postnatal mice with DNA constructs encoding fluorescent proteins. Small clusters of fluorescently labeled RGC projections to the dLGN and SC reproduced similar projection patterns as previous studies using RGC labeling with lipophilic dyes, indicating that electroporation did not interfere with normal RGC axon arbor refinement. We have utilized this protocol to analyze the r.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dumont #5 Forceps	Fine Science Tools	11252-20
Electrical Stimulator	Grass Technologies	Model S4
Oscilloscope	Agilent Technologies	Model 54621A
Audio monitor	Grass Technologies	Model AM8B
Puller	Sutter Instrument Co.	Model P-97
Vannas Scissors a	World Precision Instruments, Inc.	14003
Micro Scissors b	Ted Pella, Inc.	1347
Dumont AA Forceps c	Fine Science Tools	11210-20
Nanoinject II System	Drummond Scientific	3-000-204
Glass Pipettes	Drummond Scientific	3-000-203-G/X
Foot pedal	Drummond Scientific	3-000-026
Mineral Oil	Sigma-Aldrich	M3516
DiI	Invitrogen	D-383
N,N-Dimethylformamide	Sigma-Aldrich	D4551