Single Cell Electroporation in vivo within the Intact Developing Brain

Summary

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

Abstract

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Protocol

Equipment set-up:

1. The electrical equipment required for this technique are a stimulator, an oscilloscope, and a micropipette holder fitted with a silver wire to be inserted into the micropipette. We typically use an Axon instruments Axoporator 800A stimulator, however other common stimulators such as the Grass instruments SD9 stimulators are also suitable for single-cell electroporation. The stimulator is connected to the headstage, which interfaces with the silver wire electrode to be placed inside the glass micropipette containing an internal conducting solution. The other lead from the stimulator is connected to the oscilloscope input, which also receives....

Discussion

Single-cell electroporation (SCE) is a powerful tool for determining gene function and performing targeted genetic manipulation. The transparency of the albino tadpole and the accessibility of the brain make this model system ideally suited for visualizing neuronal growth and intracellular events within a live organism. SCE allows the visualization of the growth of a single neuron, and to perform cell-autonomous manipulations within an otherwise unaltered brain. While this video article demonstrates the procedure for sin.......