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.
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.
Equipment set-up:
Fabrication of micropipettes:
Preparation of appropriate micropipettes is a critical step in this protocol. The difficulty lies in balancing a narrow tip (less than 1μm in diameter) with one that will not easily break when puncturing the tissue. We have also found that tips with a taper angle of greater than 10 degrees are preferable.
Single-cell electroporation protocol:
For new users, it is advisable to begin with fluorescent dextran dyes since, unlike genetically encoded fluorescent proteins, they can be immediately seen under epifluorescence. Use of fluorescent dyes enables the user to directly see the location of the micropipette tip within the tissue, and upon electroporation gives instant feedback as to whether the equipment has been properly set up and whether the micropipette tip is appropriate.
Screening for successfully electroporated cells:
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 single-cell electroporation in Xenopus laevis tadpoles, this technique has been used in other organisms, and has also been used in hippocampal slices and dissociated cell cultures.
The authors thank Sharmin Hossain for the time-lapse movie capturing growth of an immature neuron and Derek Dunfield for the electron microscopy images of SCE micromicropipette tips.