Single cell electroporation in vivo within the intact developing brain.
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.
Hewapathirane
D Sesath
DS
Brain Research Centre, University of British Columbia.
Haas
Kurt
K
D000818:Animals; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry; D018274:Electroporation / Q000379:methods*; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000737:chemistry; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods; D009474:Neurons / Q000737:chemistry / Q000502:physiology*; D014982:Xenopus laevis
D005456:Fluorescent Dyes; D004247:DNA