Electroporation system for zebrafish development and genetics

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We propose to acquire an in vivo electroporation system to investigate the molecular mechanisms underlying fin regeneration in the adult zebrafish and to study zebrafish brain development and function. The zebrafish is an important model system to understand vertebrate biology and development and the accessibility of embryos, larvae, and adult tissue make it particularly amenable for gene manipulation studies by electroporation. An important goal in developmental biology and molecular genetics is to determine the function of genes.  This can be done in vivo by examining the consequences of the loss of the expression of a given gene (loss of function) or, on the contrary, its expression in a domain where it is normally not expressed (gain of function), on the normal development of the animal or the function of an organ.  In vivo electroporation is a recently developed gene transfer method to specific tissues of embryos and adult animals.  In this method, charged molecules are first microinjected in the tissue/organ of interest.  Then electric pulses are delivered to the tissue to generate the transient formation of pores in the cells membranes that allow the entry of the injected molecules.  When applied to embryos and adult animals, this technique allows the efficient introduction of DNA or other charged macromolecules into cells of an entire organ or of only a few cells, depending on the type of electrodes used.  Therefore, it does not necessarily compromise the development of the embryo or the life of the adult.  This method of gene delivery presents the advantages of being rapid, easy and cost-effective.  In zebrafish, it offers the additional advantage of allowing gene manipulation studies at all stages of the life of the animal, in contrast to microinjection alone performed in fertilized eggs which often limits the duration of the effect of the introduced macromolecule.