Electroporation of Morpholinos into single neurons of Xenopus tadpoles

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Jon Moulton
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Electroporation of Morpholinos into single neurons of Xenopus tadpoles

Bestman JE, Cline HT. The RNA binding protein CPEB regulates dendrite morphogenesis and neuronal circuit assembly in vivo. Proc Natl Acad Sci U S A. 2008 Dec 12. [Epub ahead of print]

Bestman and Cline used their in vivo single-cell electroporation protocol to introduce lissamine-tagged Morpholino antisense oligos into individual optic tectal neurons in stage 46 to 48 albino Xenopus laevis tadpoles. They knocked down expression of cytoplasmic polyadenylation element binding protein 1 (CPEB1), a component of some brain ribonucleoprotein (RNP) granules. CPEB1 binds to RNA that have cytoplasmic polyadenylation elements (CPE) in their 3’-untranslated regions; CPEB1 is estimated by bioinformatics to bind to about 7% of brain mRNAs. Binding of CPEB1 to an mRNA’s CPE represses its translation and regulates microtubule-dependant trafficking. When CPEB1 is phosphorylated, the mRNA can be polyadenylated and translated.

The authors collected time-lapse images of neurons over a period of three days after treatment with either CPEB1-targeted Morpholinos or control Morpholinos. In either treatment the dendritic arbors grew, but the arbors of neurons treated with the CPEB1-targeted Morpholinos had only about two-thirds of the total dendritic branch length of the control-treated neurons. Dendritic arbor elaboration is a hallmark of experience-dependent neural plasticity, so this observation suggests that CPEB1 activity is required for experience-based remodeling of the neural circuitry of the visual system.

Bestman JE, Ewald RC, Chiu SL, Cline HT. In vivo single-cell electroporation for transfer of DNA and macromolecules. Nat Protoc. 2006;1(3):1267-72.