Chapter 8 Vectors and Techniques for Ectopic Gene Expression in Zebrafish

Abstract

This chapter presents the methods for ectopic gene expression in zebrafish using synthetic mRNA, including microinjection instrumentation, approaches for template design, and examples of some transcription vectors presently in use. Ectopic gene expression in zebrafish can be categorized as either transient or stable, based upon the method used. Transient methods include microinjection of RNA or DNA, and are typically restricted to the studies involving the first day of development due to the limited stability of the exogenously added molecules. DNA that carries a gene of interest under promoter control is injected, and consequently this approach restricts misexpression to times after zygotic transcription has initiated. An alternative strategy for alleviating mosaicism for tissue-specific ectopic gene expression is through stable expression from a transgene, although to date only reporter molecules have been targeted in a tissue-specific manner. The chapter also discusses the design of a dissecting fluorescent microscope system that can be readily adapted at modest cost for the detection in living embryos of the green fluorescent protein (GFP) for the analysis of injection success as well as for cell lineage studies.