ImPaqT - A Golden Gate-based Immunological Toolkit for Zebrafish Transgenesis

Transgenic animals continue to play an essential role in many aspects of zebrafish research, including the development of disease models. The most widely used system for zebrafish transgenesis is the Tol2 transposon system. Here, we have developed ImPaqT ( Im munological toolkit for Paq CI-based Golden Gate Assembly of Tol2 T ransgenes), a new Tol2-based transgenesis system that utilizes Golden Gate assembly to facilitate the production of transgenic zebrafish lines. This system allows for rapid assembly of multiple fragments into a single transgene, facile swapping of individual sequences to generate new transgenes and an easy cloning workflow to incorporate new genetic elements into the existing kit. Within this toolkit framework, we have generated a number of immunological reagents to enable gene expression within major immune lineages, an array of best-in-class fluorescent proteins to visualize cell populations and transgenes as well as tools to simplify genetic manipulation, purification and ablation of these cell types. Unlike recombination-based systems, the Golden Gate approach is also expandable, allowing the incorporation of complex designs such as multi-fragment promoters within the established modular framework of ImPaqT. Here, we demonstrate the power of this new system by generating a number of novel transgenic immune reporter lines. While our toolkit has focused on the immune system as an emerging area of study within zebrafish research, the ImPaqT approach can be broadly adapted to the construction of almost any zebrafish transgene, offering new tools for the generation of transgenes within the zebrafish community.