Manipulating gene expression in the zebrafish

<jats:title>Abstract</jats:title> <jats:p>The development of techniques to introduce and express recombinant DNA constructs is an important step in the establishment of any genetic model system. Such constructs can be used to express proteins transiently during development or to produce transgenic lines that express the construct in subsequent generations. Transformation allows one to assay the effects of expressing molecules in particular embryonic domains or at different time points during development. In addition, transgene constructs that express marker genes such as the green fluorescent protein (GFP) or IacZ under the control tissue of specific enhancers lead to the marking of particular cell types and greatly simplify the observation of these cells during morphogenesis (1). Such reporter constructs can also be used to identify cis-acting sequences and, in turn, the transacting factors that regulate tissue-specific transcription. Finally, given the great number of mutants that are available to the zebrafish community, one very important use for transformation will be in the rescue of embryonic phenotypes by the microinjection of candidate genomic regions or cDNAs. It is therefore likely that transformation techniques will play an increasingly important role in zebrafish research. The aim of this chapter is to describe the methods most commonly used to transform zebrafish embryos, complete with experimental procedures and full descriptions of the equipment used. We will also cover, albeit briefly, the recently developed morpholino knockdown technique that has revolutionized zebrafish research. As well as providing protocols, we will explain why particular methods are used and what one can expect while following them.</jats:p>