Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development

We present a new protocol for zebrafish brain electroporation. Using a simple set-up with fixed spaced electrodes and microinjection equipment, it is possible to electroporate 50 to 100 embryos in 1 hour with no lethality and consistently high levels of transgene expression in numerous cells. Transfected cells in the zebrafish brain are amenable to in vivo time lapse imaging. Explants containing transfected neurons can be cultured for in vitro analysis. We also present a simple enzymatic method to isolate whole brains from fixed zebrafish for immunocytochemistry.Building on previously described methods, we have optimized several parameters to allow for highly efficient unilateral or bilateral transgenesis of a large number of cells in the zebrafish brain. This method is simple and provides consistently high levels of transgenesis for large numbers of embryos.Electroporation has been used successfully in chick embryos to perform gain of function (overexpression) and loss of function (dominant negative, small interfering RNA, morpholino) studies in various tissues, particularly the spinal cord [1,2]. More recently, similar protocols have been presented for use with Xenopus [3] and zebrafish [4-7], and somewhat more arduous technical methods can be used for in utero electroporation of mice [8,9]. All electroporation techniques are based on the application of an electric field to a tissue in the presence of a macromolecule of interest. The field induces transient pores in the plasma membrane of cells, as well as bulk flow of charged molecules toward one of the electrodes (for example, toward the cathode for negatively charged nucleic acids). This directional aspect of electroporation has been taken advantage of to unilaterally transfect the neural tube of chick, Xenopus, and zebrafish.We began experimenting with electroporation of zebrafish in order to examine the development of commissural axon projections in the brain. Unilateral electroporation is an ideal technique