Single-cell electroporation of Xenopus tadpole tectal neurons

Abstract

Single-cell electroporation (SCE) is a versatile technique for delivering electrically charged macromolecules, including DNA, RNA, synthetic oligonucleotides, peptides, dyes, and drugs, to individual cells within intact tissues. Here, we describe methods for SCE of single tectal neurons within the albino Xenopus laevis tadpole for delivery of plasmid DNA-expressing protein fluorophores or fluorescent dye. Individual neurons labeled by this technique can then be imaged in three dimensions (3D) within the intact and living brain using in vivo two-photon microscopy for studies of morphology and growth. The SCE protocol is relatively simple and requires minimal and common laboratory equipment, including a fluorescent stereomicroscope, micropipette puller, and electrical stimulator. Once equipment is set up, learning to label cells with fluorescent dyes is straightforward and usually quickly achieved, because direct visualization with fluorescent microscopy offers immediate feedback of success. The main challenges are positioning the pipette tip in a cell body layer and optimizing pipette tip shape and stimulation parameters. Once fluorescent dye loading has been achieved, transfecting neurons with DNA should follow by using the same pipette tip parameters, but extending the stimulation parameters, because plasmid DNA is larger than dye and requires formation of larger pores. Detectable expression of protein fluorophores from transfected DNA typically takes 6-12 h. SCE for dye loading or DNA transfection of tadpole tectal neurons is highly efficient and can be learned in 1 or 2 d by novice laboratory personnel.