Brain Development

Jennifer E. Bestman, Hollis T. Cline

Antisense morpholino oligonucleotides (MOs) have become a valuable method to knock down protein levels, to block mRNA splicing, and to interfere with miRNA function. MOs are widely used to alter gene expression during development of Xenopus and zebra fish, where they are typically injected into the fertilized egg or blastomeres. Here, we present methods to use electroporation to target delivery of MOs to the central nervous system of Xenopus laevis or Xenopus tropicalis tadpoles. Briefly, MO electroporation is accomplished by injecting MO solution into the brain ventricle and driving the MOs into cells in the brain with current passing between two platinum plate electrodes, positioned on either side of the target brain area. The method is straightforward and uses standard equipment found in many neuroscience labs. A major advantage of electroporation is that it allows spatial and temporal control of MO delivery and therefore knockdown. Co-electroporation of MOs with cell-type specific fluorescent protein expression plasmids allows morphological analysis of cellular phenotypes. Furthermore, co-electroporation of MOs with rescuing plasmids allows assessment of specificity of the knockdown and phenotypic outcome. By combining MO-mediated manipulations with sophisticated assays of neuronal function, such as electrophysiological recording, behavioral assays, or in vivo time-lapse imaging of neuronal development, the functions of specific proteins and miRNAs within the developing nervous system can be elucidated. These methods can be adapted to apply antisense morpholinos to study protein and RNA function in a variety of complex tissues.