Developing Time Resolved Genetics in Zebrafish

Scott A Kera, Pace University Suneel Agerwala , Pace University Jack H Horne, Pace University

Abstract Identifying the genes necessary for specific steps in the development of the nervous system is fundamental to molecular and developmental neuroscience. Loss-of-function analysis in model organisms has yielded a wealth of information on which molecules are important for various aspects of neuronal development. However, the vast majority of these studies have analyzed individuals for which the function of the gene being studied was absent even at the very early stages of development, which poses a significant problem for genes that function both in early development, and at later steps in neuronal development. Here we begin to develop a loss-of-function method using in vivo electroporation in Zebrafish (Danio rerio) that can be initiated at specific stages of development. The efficacy, viability, and temporal resolution of in vivo electroporation was assessed by incorporation of GFP-expression plasmids and in vivo fluorescence imaging. We show that electroporation can target multiple neuronal populations of developing neurons including midbrain, hindbrain, cerebellum, and neural retina. We find that voltages that lead to efficient, reproducible GFP expression are well within the range for which there is very high viability. GFP expression can be observed within 8 hours of electroporation, and reaches maximal expression by 24 hours. The efficacy and timecourse of electroporation suggests that this method could be used for time-resolved loss-of-function analysis by co-electroporation of GFP expression plasmid with morpholino anti-sense oligonucleotides.

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