Generating new transgenic lines is an ongoing focus of the lab, allowing us to investigate and manipulated increasingly more specific neuronal populations. In our fish room we have available a range of different zebrafish lines, which allow us to, for example, label distinct neuronal cell types, observe the effects of mutations, visualise calcium activity, and optogenetically manipulate neuronal activity. Most of the lines are generated by plasmid microinjection at the one cell stage in the laboratories of collaborators or in our own laboratory. We use a variety of different genetic techniques to generate the needed zebrafish lines, with the most common approach being a combination of Tol2 mediated insertion of DNA into the genome and the Gal4-UAS system.
The Gal4-UAS system is a flexible and efficient way to quickly generate many zebrafish with novel properties through the use of a small number of starter lines. Briefly, a Gal4 expressing promoter line must be crossed with a UAS containing reporter line in order for expression to occur. Once established, maintaining a diverse range of Gal4 promoter lines and UAS reporter lines – which can be crossed to generate the lines needed for an experiment – takes less time than that needed to inject a full plasmid containing both inserts into individual embryos, raise these embryos to adulthood (F0) and use their offspring (F1).