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One caveat to current loss-of-function approaches in zebrafish is that they typically disrupt gene function from the beginning of development. This can be problematic when attempting to study later developmental events. In vivo electroporation is a method that has been shown to be effective at incorporating reagents into the developing nervous system at multiple later developmental stages. The temporal and spatial characteristics of in vivo electroporation that have been previously demonstrated suggest that this could be a powerful approach for time-resolved loss-of-function analysis. Here, in an attempt to demonstrate the efficacy of this approach for analysis of a specific developmental timeframe--that of initial development of the zebrafish visual system-we have done a systematic characterization of the efficiency of in vivo electroporation in zebrafish across multiple developmental stages, from 24 to 96 h postfertilization. We show that electroporation is efficient at delivering expression plasmids to large numbers of neurons at multiple developmental steps, including 24, 48, or 96 h postfertilization. Expression from electroporated plasmids is maximal within 24 h, and significant and useful expression is seen within 6 h. Electroporation can be used to deliver two separate expression plasmids (green fluorescent protein and mCherry), resulting in coexpression in 97% of cells. Most importantly, electroporation can be used to incorporate siRNA reagents, resulting in 84% knockdown of a target protein (green fluorescent protein). In conclusion, in vivo electroporation is an effective method for delivering both DNA-based expression plasmids and RNA interference-based loss-of-function reagents, and exhibits the appropriate characteristics to be useful as a time-resolved genetic approach to investigate the molecular mechanisms of visual system development.

The Temporal Resolution ofIn VivoElectroporation in Zebrafish: A Method for Time-Resolved Loss of Function